PLANT PATHOGEN

INTERACTION:
RECENT TRENDS

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ARINDAM BARMAN
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Soil Microbes and their interaction with plants

CHAPTER
Soil Microbes and their
1 interaction with plants
--------------------------------------------------------------------------
Roshan Kumar, Vivek Prakash Pankaj, Avijit Tarafdar, Koushik
Biswas and Shailendra Kumar

1. Introduction
This chapter will look forward at different types of microorganism’s interaction
with Plants. It will also discuss the effects which will arise after the interaction of mi-
crobes with plants. Plant-Microbe Interactions Encounters between plant cells and both
‘friendly’ and ‘hostile’ microbes (such as those in symbiotic and pathogenic interac-
tions, respectively) trigger a range of highly dynamic plant cellular responses. Densely
colonized soil contains beneficial mycorrhizal fungi and rhizobia, which associate
with roots and provide plants with mineral nutrients and fixed nitrogen, respectively,
in exchange for carbon. By contrast, plants are constantly exposed to a range of
fungal, bacterial and viral pathogens, and have evolved unique defense mechanisms
to fight these infections. These include reorganization of the cytoskeleton, organelle
translocation, vesicle trafficking, and alterations in subcellular protein localization. Re-
cent progress in this border-land that bridges the fields of plant–microbe interac-
tions and cell biology heralds the transition from descriptive phenomenology to
the identification and characterization of key molecules that are involved in these
processes. Intriguingly, molecular events that occur in plant cells in response to
microbes also take place upon abiotic wounding and during fundamental plant
developmental processes, such as the tip growth of pollen, root hairs and trichomes.
Thus, elementary ‘activity modules’ that are required for the generation of cell polar-
ity in plant morphogenesis appear to be re-used in both abiotic and biotic stress
response pathways.
1.1 Dynamic Subcellular Responses in Plants

i. Host cell pre-alert

Chemistry is the language of cells, and plant interacting microbes are no excep-
tion. Any plant microbe interaction with chemical signaling, on investigation revealed
the existence of a precise molecular dialogue prior to and during direct cellto-cell con-
tact [Geurts et al. 2005]. Pathogenic fungal effector molecules are reported to trigger

1
 plant- pathogen interaction

signaling cascades [Zhang and Klessig, 2001], gene activation [Schenk et al. 2000],
the release of defense-related molecules, and systemic reactions. In symbioses, root
nodulation is the most-studied system from this point of view [Gage, 2010]. Isolated
chemical signals released by rhizobia, the so-called Nod factors, induce a number
of rapid molecular or cellular responses in root hairs of compatible plant spe-
cies, including the activation of specific intracellular signaling pathways leading to
gene activation [Lohar et al. 2006]. Additional later responses to Nod factors include
the initiation of nodule organogenesis in the inner cortex. In the early 1990’s, Heids-
tra and coworkers (1994) described the swelling of roothair tips exposed to a Nod
factor solution, later correlated with a transient fragmentation and reorganization
of the apical cytoskeleton [Cárdenas et al. 1998]. Recently, it has been shown that
exogenous application of Nod factors to root hairs results in the rapid fragmentation
of actin bundles and is accompanied by increased apical influxes and intracellular
levels of calcium.

ii. Direct cell contact

Plant non-host resistance strategies represent the first line of defense against
invading microbes [Miwa et al. 2006]. Surveillance system that allows plants to
recognize and effectively respond to most ‘wanna-be’ pathogens, a phenomenon
frequently termed ‘non-host resistance’. Despite the fundamentally different final
outcome of microbial contacts, plant cells exhibit a surprisingly similar set of highly
dynamic cellular responses during contacts. These activities ultimately lead to ex-
tensive polarization towards the microbe at the single-cell level [Schmelzer, 2002].
Among subcellular responses, the 1st visible reaction is rapid translocation of
the cytosol and subcellular elements to the contact site. This is called cytoplasmic
aggregation (CA) [Heath et al. 1997]. CA results from the establishment of a strik-
ing asymmetry within the cell [Schmelzer, 2002] through rapid site-directed cyto-
plasmic streaming [Heath et al. 1997]. CA depends primarily on the action of the actin
cytoskeleton. Recent cytochemical and GFP-labeling of actin microfilaments have
revealed their radial organization around the penetration site in many plant-pathogen
interactions ; while microtubules show more heterogeneous patterns in barley and
flax , in Soybean, in Arabidopsis. Most organelles assemble at the potential infec-
tion site, where they contribute to an intense secretory activity , reinforcing the wall
with compounds that improve its resistance to the chemical and physical attack
directed by the pathogen [Zeyen et al. 2002]. Cell-wall appositions, or papillae,
develop around and in front of fungal appressoria, intercellular hyphae, and pene-
tration pegs [Zeyen et al. 2002]. Secretion of antifungal compounds has been proposed
by Stein and colleagues (2006). When cell-wall appositions are not sufficient to
arrest noncompatible pathogens or their effectors from reaching the cytoplasm, a
second line of defense can be initiated. Specific receptors trigger signaling cascades
that program cell death [O’Connell and Panstruga 2006].
2
Soil Microbes and their interaction with plants

1.2 Comparative analysis of host cell responses

i. Host vesicle transport and secretion

Members of the superfamily of soluble Nethylmaleimide- sensitive factor

adaptor protein receptor (SNARE) polypeptides contribute to limiting host cell en-
try by powdery mildew fungi both in monocot and dicot plant species. Mutations
in the gene encoding plasma membrane (PM)-resident Arabidopsis AtPEN1 target
membrane SNARE (t-SNARE, also referred to as syntaxin) allow enhanced cell
invasion by the grass powdery mildew fungus Blumeria graminis f. sp. hordei
(Bgh) ; suggesting that t-SNARE function is essential to arrest fungal ingress at the cell
periphery [Schulze, 2004]. Further SNARE protein, SNAP-25, is known to form bi-
nary complexes with syntaxins to promote membrane-fusion events in animal and
yeast cells. Indirect evidence for a role of exocytosis in non-host resistance - Se-
cretion of a cocktail of metabolites that have antimicrobial activity confers tissue-
specific resistance to a range of bacterial microbes in the roots of Arabidopsis.
Activation of the secretory pathway represents systemic acquired resistance (SAR),
a plant-wide immune response that is triggered upon a local stimulation of the
plant’s pathogen surveillance system.

ii. Reorganization of the plant cytoskeleton and organelle positioning

Numerous pharmacological inhibitor studies have demonstrated the importance

of cytoskeletal rearrangements for the execution of pathogen entry control at the cell
periphery. Actin microfilaments play a pivotal role in timely and spatial recruitment
of the plant’s defensive forces at infection sites [30]. Dynamics of cytoskeleton and
endomembrane reorganization during pathogen attack have been explored in living
cells through studies of GFP-tagged cell components. E.g., extensive changes in
the distribution and morphology of the ER followed during a 15-minute period
from the onset of reorganization beneath a Phytophthora sojaehypha growing
across the outer epidermal cell wall [Hardham et al. 2008]. Although experiments
corroborate the importance of adaptive cytoskeleton remodelling in potential patho-
gens, molecules that signal and carry out dramatic reorganizations are still elusive.
Possible role for RAC/ROP family G proteins: potent regulators of pathogen-
induced plant microfilament reassembly that might be targeted by fungal pathogens
to establish compatibility. Another plant susceptibility factor, barley MLO protein,
also affects actin-dependent control of pathogen entry at the cell periphery.

iii. Dynamic changes in sub-cellular protein localization

Identification of individual proteins that exhibit analtered subcellular localiza-

tion upon a biotic stimulus is a fresh finding. Application of a peptide elicitor,
Pep13, derived from the oomycete pathogen Phythophtora triggers an innate im-
3
 plant- pathogen interaction

mune response in cultured parsley cells that involves the activation of three mitogen
activated protein kinases (MAPKs); immunolabelling, all 3 MAPKs exhibits an
increase in nuclear localization upon activation [Rodriguez et al. 2006].

iv. Polarizing the host cell

Hypotheses postulated concerning the way a cell perceives contact with a po-
tential guest. Hypothetical mechanisms proposed that involve organization of spe-
cialized membrane domains, which label the contact site, and focal accumulation
of membrane-bound proteins has been demonstrated by yellow fluorescent protein
tagging [Bhat et al. 2005].

1.3 Cytoskeleton and Cellular Response Targets

I. Cytoskeleton functions: In plant–microbe interactions Plant cytoskeleton con-

tributes to establishment of cell polarity during plant development and morpho-
genesis. Rapid changes in regular cytoskeleton architecture occur upon contact
of individual plant cells with both pathogenic and symbiotic microbes. In case
of pathogens, polarized cytoskeletal rearrangements are thought to allow local-
ized delivery of cargo for defense execution, while in symbiotic interactions the
reorganization may advance establishment of the symbiotic relationship. Microbial
metabolites and effector proteins are released into plant cells for manipulation of the
host cytoskeleton, while some secreted plant defensive polypeptides may target the
microbial cytoskeleton. The cytoskeleton thus emerges as a potential mutual target
in plant–pathogen combats that appears to be under attack by effector molecules from
both sides [Altenbach and Robatzek, 2007].

II. Regulator and target of biotic interactions in plants: Plant cytoskeleton

composed of microtubules and actin microfilaments, plays role in plant cell growth
and development and intracellular organization and motility.

a. Response to pathogenic fungi and oomycetes

Cytoplasmic aggregation is an example of site directed cytoplasmic stream-

ing and is dependent upon the action of the actin component of the cytoskeleton,
shown using inhibitors of actin polymerization [Diaz et al. 1996]. Assessment of
the contribution made by cytoplasmic aggregation to plant resistance may be com-
plicated by an influence of the pathogen on host cell structure and metabolism.
As well as redirection by the plant for defense purposes, redeployment of plant
materials via cytoskeletal rearrangements could also be orchestrated by an invad-
ing pathogen as part of its strategy to obtain nutrients from the plant.

4
Soil Microbes and their interaction with plants

b. Plant cytoskeletal response to mycorrhizal fungi

Two main categories of mycorrhizal symbiosis: ectomycorrhizal and endo-

mycorrhizal associations. Ectomycorrhizal fungi do not penetrate host cell wall
but form a dense layer, called the mantle, on surface of the root and a network
of intercellular hyphae within the root tissues. In endomycorrhizae, fungal cells
penetrate the plant cell wall and elaborate specialized infection structures within
the plant cell; although, they remain surrounded and separated from the host
cytoplasm by an intact plant plasma membrane. Increases in expression of tubulin
genes and concentrations of plant a-, b-, and g-tubulins and actin have been observed
in a variety of ecto- and endomycorrhizas [Bonfante et al. 1996].

c. Plant cytoskeleton targeted by pathogenic bacteria

Rhizobia and many plant and animal bacterial pathogens possess the type-III
secretion system that injects bacterial proteins (effectors) into the host cytoplasm.
In animals, the host cytoskeleton, in particular actin microfilaments, is a major tar-
get of type-III effectors for pathogen virulence. Various type-III effectors of plant
pathogenic bacteria are virulence factors that suppress plant defense responses
such as hypersensitive cell death and expression of defense genes [Hauck et al.
2006]. Till date, no reports of type III effectors targeting plant cytoskeletal ele-
ments exist.

1.4 Molecular Characterization and Functional Analysis

Analysis of the fully sequenced genome of the wheat leaf-specific fun-

gal pathogen Mycosphaerella graminicola identified only a single gene encoding a
member of the necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein family
(NLP). NLP proteins have frequently been shown to trigger cell death and activa-
tion of defense signaling reactions in dicotyledonous plants. However, complete
lossof-function reverse genetics analyses for their importance in the virulence of
eukaryotic plant pathogens are generally lacking. Real-time quantitative polymerase
chain reaction on MgNLP demonstrated the gene to be specifically expressed in
planta. Peak expression was observed during the immediate presymptomatic phase
of colonization of a susceptible host genotype. This was followed by a dramatic
decrease during disease lesion formation which, in this system, exhibits character-
istics of host programmed cell death (PCD). No comparable peak in transcript
levels was seen during an incompatible interaction with a host genotype exhibiting
gene-for-gene–based disease resistance. Heterologously expressed MgNLP protein
induced necrotic cell death and the activation of defense-related genes when in-
filtrated into Arabidopsis leaves but not in leaves of a susceptible wheat genotype.
MgNLP infiltration also failed to stimulate wheat mitogenactivated protein kinase

5
 plant- pathogen interaction

activities. Finally, targeted deletion of M. graminicola MgNLP caused no detect-

able reduction in plant pathogenicity or virulence, suggesting that this protein is
not a major virulence determinant during fungal infection of its host plant. This
represents the first complete loss-of-function analysis of NLP in a eukaryotic plant
pathogen and findings in the context of possible functions for NLP in pathogens
which only infect monocotyledonous plants [Chisholm et al. 2006].

1.5 Root hair response

Symbiotic relationship with rhizobia Changes in cytoskeletal arrays in root

hairs and cortical cells occur during the establishment of a symbiotic relationship with
certain gram-negative bacteria collectively called rhizobia, and it is clear that micro-
tubules and actin microfilaments play active and necessary roles in root hair
curling, growth of the infection thread and root nodule development. Root hairs fol-
lowing inoculation Rhizobial attachment, or application of hostspecific nodulation
(Nod) factors causes a localized influx of calcium, depolarization of the plant plasma
membrane, alkalinization of the cytoplasm, and curling (or deformation) of the
hair. Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family
that is abundantly transcribed in leaves and nodules. Inoculation with Rhizobium
sp. NGR234, which nodulates V. unguiculata, or with NGRDnodABC, a mutant de-
ficient in Nod factor production, induced rapid accumulation of CHS mRNAs in
roots and root hairs. As both Nod+ and Nod– bacteria provoke responses, induction
of CHS gene expression may involve symbiotic or defense responses. Four days
after inoculation with the wild-type Rhizobium sp., the transcript levels increased
in roots but decreased in root hairs. Use of a region unique to the 5¢ end of a specific
CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24
h after inoculation with both rhizobia were specific to this gene. Transcripts of this
gene in roots were only detectable 4 days after treatment with NGR234. It is possible
therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia
entering legume roots. Purified Nod factors induced accumulation of transcripts,
showing that they might be part of the signal transduction pathway leading to CHS
expression. Response of the actin cytoskeleton in vetch root hairs after application
of host-specific Nod factor is studied. Within 3 to 15 min, the number of sub-apical fine
bundles of actin filaments (FB-actin) increased in all developmental stages. Tip
growth resumed only in hairs in which the FB-actin density and the length of the
region with FB-actin exceeded a minimal value. If the fine bundles of actin mi-
crofilaments in the tip of the hair are depolymerized by treatment with cytochalasin
D, deformation but not new outgrowth occurs. These results are consistent with a role
of the subapical actin microfilaments in targeting vesicle exocytosis to the growing
tip of the hair [Norbert et al. 1999].

6
Soil Microbes and their interaction with plants

1. The plant immune system

Plant pathogens use diverse life strategies. Pathogenic bacteria proliferate in
intercellular spaces (the apoplast) after entering through gas or water pores (stomata
and hydathodes, respectively), or gain access via wounds. Nematodes and aphids feed
by inserting a stylet directly into a plant cell. Fungi can directly enter plant epidermal
cells, or extend hyphae on top of, between, or through plant cells. Pathogenic and
symbiotic fungi and oomycetes can invaginate feeding structures (haustoria), into the
host cell plasma membrane. Haustorial plasma membranes, the extracellular matrix,
and host plasma membranes form an intimate interface at which the outcome of the
interaction is determined. These diverse pathogen classes all deliver effector molecules
(virulence factors) into the plant cell to enhance microbial [Link], unlike mam-
mals, lack mobile defender cells and a somatic adaptive immune system. Instead, they
rely on the innate immunity of each cell and on systemic signals emanating from infec-
tion sites1–3.
There are two branches of the plant immune system. One uses trans membrane
pattern recognition receptors (PRRs) that respond to slowly evolving microbial- or-
pathogen associated molecular patterns (MAMPS or PAMPs), such as flagellin6. The
second acts largely inside the cell, using the polymorphic NB-LRR protein products
encoded by most R genes. They are named after their characteristic nucleotide binding
(NB) and leucine rich repeat (LRR) domains. NB-LRR proteins are broadly related
to animal CATERPILLER/NOD/NLR proteins and STANDATPases. Pathogen effec-
tors from diverse kingdoms are recognized by NB-LRR proteins, and activate similar
defence responses. NBLRR-mediated disease resistance is effective against pathogens
thatcan grow only on living host tissue (obligate biotrophs), or hemi biotrophic patho-
gens, but not against pathogens that kill host tissue during colonization (necrotrophs)
[Glazebrook, 2005]. Our current view of the plant immune system can be represented
as a four phased ‘zigzag’ model (Fig. 1), in which we introduce several important ab-
breviations. In phase 1, PAMPs (or MAMPs) are recognized by PRRs, resulting in
PAMP-triggered immunity (PTI) that canhalt further colonization. In phase 2, success-
ful pathogens deploy effectors that contribute to pathogen virulence. Effectors can in-
terfere with PTI. This results in effector-triggered susceptibility (ETS).
In phase 3, a given effector is ‘specifically recognized’ by one of the NB-LRR
proteins, resulting in effector-triggered immunity (ETI).Recognition is either indirect,
or through direct NB-LRR recognition of an effector. ETI is an accelerated and ampli-
fied PTI response, resulting in disease resistance and, usually, a hypersensitive cell
death response (HR) at the infection site. In phase 4, natural selection drives pathogens
to avoid ETI either by shedding or diversifying the recognized effector gene, or by
acquiring additional effectors that suppress ETI. Natural selection results in new R
specificities so that

7
 plant- pathogen interaction

Figure 1. A zigzag model illustrates the quantitative output of the plant immune sys-
tem.

In this scheme, the ultimate amplitude of disease resistance or susceptibility

is proportional to [PTI – ETS1ETI]. In phase 1, plants detect microbial/pathogen-
associated molecular patterns (MAMPs/PAMPs, red diamonds) via PRRs to trigger
PAMP-triggered immunity (PTI). In phase 2, successful pathogens deliver effectors
that interfere withPTI, or otherwise enable pathogen nutrition and dispersal, resulting
ineffector-triggered susceptibility (ETS). In phase 3, one effector (indicated in red) is
recognized by an NB-LRR protein, activating effector-triggered immunity (ETI), an
amplified version of PTI that often passes a threshold for induction of hypersensitive
cell death (HR). In phase 4, pathogen isolates are selected that have lost the red effec-
tor, and perhaps gained new effectors through horizontal gene flow (in blue)—these
can help pathogens to suppress ETI. Selection favours new plant NB-LRR alleles that
can recognizeone of the newly acquired effectors, resulting again in ETI ETI can be
triggered again. Below, we review each phase in turn, we update the experimental vali-
dation of the ‘guard hypothesis’, and we consider future challenges in understanding
and manipulating the plant immune system. We will not discuss the small RNA-based
plant immune system active against viruses [Voinnet, 2005] or the active response of
plants to herbivores [Kessler & Baldwin, 2002].

2.1 Microbial patterns and plant pattern recognition

Basal disease resistance is activated by virulent pathogens on susceptible hosts.

Thus, basal disease resistance is, at first glance, PTI minus the effects of ETS; how-
ever, there is also likely to be weak ETI triggered by weak recognition of effectors,

8
Soil Microbes and their interaction with plants

as detailed below. Hence, the most accurate definition of basal defence wouldbe ‘PTI
plus weak ETI, minus ETS’. The archetypal elicitor of PTI isbacterial flagellin, which
triggers defence responses in various plants [Gomez-Gomez, 2002]. Flagellum-based
motility is important for bacterial pathogenicity in plants6. A synthetic 22-amino-acid
peptide (flg22) from aconserved flagellin domain is sufficient to induce many cellu-
lar responses[Felix et al. 1999] including the rapid (,1 h) transcriptional induction of
atleast 1,100 Arabidopsis thaliana (hereafter Arabidopsis) genes. Agenetic screen using
flg22 defined the Arabidopsis LRR-receptor kinase FLS2, which binds flg22. FLS2 and
mammalianTLR5 recognize different flagellin domains. FLS2 is internalized following
stimulation by a receptor-mediated endocytic process that presumably has regulatory
functions. fls2mutants exhibit enhanced sensitivity to spray application of pathogenic
Pseudomonas syringaepv. Tomato DC3000 (PtoDC3000), but not to syringe infiltration
into the leaf apoplast, suggesting that FLS2 acts early against pathogeninvasion.

Plant pathogenic bacteria deliver effectors per strain into host cells using type
III secretion systems (TTSS). Bacterial effectorscontribute to pathogen virulence, of-
ten by mimicking or inhibitingeukaryotic cellular functions. A pathogenic P. syringae
strainmutated in the TTSS, and unable to deliver any type III effectors, triggers a faster
and stronger transcription re-programming in bean than does the isogenic wild-type
strain [Jakobek et al. 1993]. This strain, representing the sum of all bacterial MAMPs/
PAMPs, induces transcription of essentially the same genes as flg22. Hence, the type
III effectors from any successful bacterial pathogen dampen PTI sufficiently to allow
successful colonization (Fig. 1).

2.2 Indirect and direct host recognition of pathogen effectors

Effectors that enable pathogens to overcome PTI are recognized by specific

disease resistance (R) genes. Most R genes encode NB-LRR proteins; there are, 125
in theArabidopsisCol-0 genome. If one effector is recognized by a corresponding NB-
LRR protein, ETI ensues. The recognized effector is termed an avirulence (Avr) pro-
tein. ETI is a faster and stronger version of PTI that often culminates in HR 53 (Fig. 1).
HR typically does not extend beyond the infected cell: it may retard pathogen growth in
some interactions, particularly those involving haustorial parasites, but is not always ob-
served, nor required, for ETI. It is unclear what actually stops pathogen growth in most
cases. Very little is known about the signalling events required to activate NB-LRR-
mediated ETI. NB-LRR proteins are probably folded in a signal competent state by
cytosolic heat shock protein and other receptor co-chaperones [Schulze-Lefert, 2004,
Holt et al. 2005]. The LRRs seem to act as negative regulators that block inappropriate
NB activation. NB-LRR activation involves intra- and intermolecular conformational

9
 plant- pathogen interaction

changes and may resemble the induced proximity mechanism by which the related ani-
mal Apaf-1 protein activates programmed cell death [Takken et al. 2006]. NB-LRR ac-
tivation results in a network of cross-talk between response pathways deployed, in part,
to differentiate biotrophic from necrotrophic pathogen attack. This is maintained by the
balance between salicylic acid, a local and systemic signal for resistance against many
biotrophs, and the combination of jasmonic acid and ethylene accumulation as signals
that promote defence against necrotrophs. Additional plant hormones are likely to alter
the salicylicacid–jasmonic-acid/ethylene signalling balance. Arabidopsis mutants de-
fective in salicylic acid biosynthesis or responsiveness are compromised in both basal
defence and systemic acquired resistance (SAR). NB-LRR activation induces differ-
ential salicylic-acid- and ROSdependent responses at and surrounding infection sites,
and systemically [Dorey et al. 1997]. The NADPH-oxidase-dependent oxidative burst
that accompanies ETI represses salicylic acid-dependent cell death spread in cells sur-
rounding infection sites. Local and systemic changes in gene expression are mediated
largely by transcription factors of the WRKY and TGA families.

Several NB-LRR proteins recognize type III effectors indirectly, by detecting

products of their action on host targets, consistent with the ‘guard hypothesis’ [Dangl
et al. 2001]. The key tenets of this hypothesis are that: (1) an effector acting as a viru-
lence factor has a target(s) in the host; (2) by manipulating or altering this target(s) the
effector contributes to pathogen success in susceptible host genotypes; and (3) effec-
tor perturbation of a host target generates a ‘pathogen-induced modifiedself’ molecu-
lar pattern, which activates the corresponding NB-LRR protein, leading to ETI. Three
important consequences of this model, now supported by experimental evidence, are
that: (1) multiple effectors could evolve independently to manipulate the same host
target, (2) this could drive the evolution of more than one NB-LRR protein associ-
ated with a target of multiple effectors, and (3) these NB-LRRs would be activated
by recognition of different modified-self patterns produced on the same target by the
action of the effectors in (1). RIN4, a 211-amino-acid, acylated [Kim et al. 2005] and
plasma-membrane-associated protein, is an archetypal example of a host target of type
III effectors that is guarded by NB-LRR proteins (Fig. 2). It is manipulated by three dif-
ferent bacterial effectors, and associatesin vivowith two ArabidopsisNB-LRR proteins
(Fig. 2a and 2b). Two unrelated type III effectors, AvrRpm1 and AvrB, interact with
and induce phosphorylation of RIN4. This RIN4 modification is predicted to activate
the RPM1 NB-LRR protein. A third effector, AvrRpt2 is a cysteine protease, activated
inside the host cell , that eliminates RIN4 by cleaving it at two sites. Cleavage of RIN4
activates the RPS2 NB-LRR protein. Activation of both RPM1 and RPS2 requires the
GPI-anchored NDR1 protein, and RIN4 interacts with NDR1. If RIN4 was the only tar-
get for these three effectors, then its elimination would abolish their ability to add viru-
10
Soil Microbes and their interaction with plants

lence to a weakly pathogenic strain. However, elimination of RIN4 demonstrated that

it is not the only host target for AvrRpm1 or AvrRpt2 in susceptible (rin4 rpm1 rps2)
plants. Additionally, AvrRpt2 can cleavein vitroseveral Arabidopsisproteins that con-
tain its consensus cleavage site. Hence, any effector’s contribution to virulence might
involve manipulation of several host targets, and the generation of several modified-
self molecules. However, the perturbation of only one target is sufficient for NB-LRR
activation. RIN4 negatively regulates RPS2 and RPM1 (and only these two NB-LRR
proteins). But what is the function of RIN4 in the absence of RPS2 and RPM1 Inrpm1
rps2 plants, AvrRpt2 or AvrRpm1 (and possibly other effectors) manipulate RIN4 (and
possibly associated proteins or other targets) in order to suppress PTI [Jia et al. 2001].
Thus, plants use NB-LRR proteins to guard against pathogens that deploy effectors
to inhibit PAMP-signalling. Additional examples of indirect recognition are detailed
in Fig. 2; these include both intra- and extra-cellular recognition of pathogeninduced
modified self. Not all NB-LRR recognition is indirect, and there are three examples of
direct Avr-NB-LRR interaction. The flax Llocus alleles encode NB-LRR proteins that
interact in yeast with the corresponding AvrL proteins, providing the first evidence that
effector diversity determining NB-LRR recognition can be correlated perfectly with
effector–NB-LRR-protein interaction. Both L and AvrL proteins are under diversify-
ing selection, arguing for a direct evolutionary arms race. The allelic diversity of other
fungal and oomycete pathogen effectors, and of their corresponding host NB-LRR pro-
teins as described above, also suggests direct interaction, though this remainsto be dem-
onstrated. The evolutionary radiation of several hundred thousand angiosperm plant
species, 140–180 million years ago was probably accompanied by many independent
cases of pathogen co-evolution, particularly of host-adapted obligate biotrophs. Most
plants resist infection by most pathogens; they are said to be ‘non-hosts’. This non-host
resistance could be mediated by at least two mechanisms. First, a pathogen’s effectors
could be ineffective on a potential new, but evolutionarily divergent, host, resulting in
little or no suppression of PTI, and failure of pathogen growth. Alternatively, one or
more of the effector complement of the would-be pathogen could be recognized by the
NB-LRR repertoire of plants other than its coadapted host, resulting in ETI. These two
scenarios predict different outcomes with respect to the timing and amplitude of the
response they would trigger, and they also give rise to different evolutionary pressures
on both host and pathogen. Non-host resistance in Arabidopsis against the non-adapted
barley pathogen, B. graminisf. sp. Hordei (Bgh) normally involves the rapid produc-
tion of cell wall appositions (physical barriers) and antimicrobial metabolites at the
site of pathogen entry, but no HR. Arabidopsispenetration (pen) mutants are partially
compromised in this response. PEN2 is a peroxisomal glucosyl hydrolase, and PEN3
encodes a plasma membrane ABC transporter. PEN2 and PEN3 are both recruited to

11
 plant- pathogen interaction

attempted fungal entry sites, apparently to mediate the polarized delivery of a toxin to
the apoplast.

.
Figure 2. Plant immune system activation by pathogen effectors that generate modi-
fied self molecular patterns.

The actin cytoskeleton probably contributes to this response [. Yun et al. 2003],
perhaps as a track for PEN2-containing peroxisomes or vesicles. This preinvasion non-
host resistance is genetically separable from a postinvasion mechanism that requires
12
Soil Microbes and their interaction with plants

additional elements that regulate both PTI and ETI. Elimination of both PEN2 and
PTI/ETI signalling transforms Arabidopsisinto a host for an evolutionarily nonadapted
fungal pathogen. This suggests that non-host resistance comprises mechanistically dis-
tinct layers of [Link] PEN1 syntaxin acts in a different pre-invasion non-host
resistance pathway. PEN1 is likely to be part of a ternary SNARE complex that secretes
vesicle cargo to the site of attempted fungal invasion, contributing to formation of
cell wall appositions. Specific seven-transmembrane MLO (mildew resistance locus
O) family members negatively regulate PEN1-dependent secretion at sites of attempted
pathogen ingress. Recessive mlomutations in eitherArabidopsisor barley result in re-
sistance to the respective coevolved powdery mildew pathogens. Hence, in both Ara-
bidopsis and barley, these fungi might suppress PEN1-mediated disease resistance by
activation of MLO. This remarkable set of findings implies that a common host cell
entry mechanism evolved in powdery mildew fungi at or before the monocot–dicot
divergence.PEN2and PEN3genes are induced by flg22, indicating that they might be
involved in PTI.

Non-host resistance can also be mediated by parallel ETI responses. For ex-
ample, four bacterial effectors from a tomato pathogen unable to colonize soybean
can each trigger specific soybean Rgenes when delivered from a soybean pathogen.
Deletion of these effector genes from the tomato pathogen diminishes its virulence on
tomato, but does not allow it to colonize soybean [Lorang et al. 1994]. Hence, there
might be other factors lacking in this strain that are required to colonize soybean. Also,
a widely distributed, monomorphic effector acting as an avirulence protein is sufficient
to renderMagnaporthe oryzaestrains unable to colonize rice. Its presence in over strains
that successfully colonize perennial ryegrass suggests a virulence function. Finally,
Arabidopsisnon-host resistance toLeptosphaeria maculans, a fungal pathogen ofBras-
sica, is actually mediated by unlinked NB-LRR proteins present in each parent of a
cross between two accessions.

Hence, cryptic NB-LRR mediated responses acting in parallel can limit patho-
gen host range. Pathogens dodge host surveillance the effectiveness of ETI selects for
microbial variants that can avoid NB-LRR-mediated recognition of a particular ef-
fector (Fig. 3). Effector allele frequencies are likely to be influenced by their mode of
action. The diversity of both flax rust AvrL alleles and oomycete Atr13 and Atr1 alleles
suggests one means of effector evolution. These proteins are likely to interact directlyin
plantawith proteins encoded by alleles of the flaxLandArabidopsis RPP1andRPP13lo-
ci, respectively. The high level of diversifying selection among these effector alleles
13
 plant- pathogen interaction

is presumably selected by host recognition, and hence acts oneffector residues that are
probably not required for effector function.

Figure 3. Co-evolution of hostRgenes and the pathogen effector complement.

3. Infection and plant defense responses during plant bacterial interaction

Among the about 7100 classified bacterial species, roughly 150 species cause
diseases to plants obtaining nutrients from these plants for their own growth by more or
less specialized mechanisms. Although found in all parts of the world, bacterial diseas-
es are mostfrequent and severe in tropical and subtropical countries, where warm and
humid conditions are ideal for bacterial growth. Indeed, consistent annual crop yield
losses are recorded in these countries. Bacterial diseases are essentially characterized
by symptoms of leaf and fruit spots, cankers, blights, vascular wilts, rots and tumors.
They are most often caused by Gram-negative bacteria belonging to the Proteobacteria
phylum, including Xanthomonadaceae, Pseudomonadaceaeand Enterobacteriaceae
families. Except for a few rare cases, phytopathogenic bacteria provoke diseases in
plants by penetrating into host tissues. Penetration occurs through natural openings,
such as stomata, hydathodes, lenticels, nectarthodes, stigma etc., or through wounds.
Bacteria colonize the apoplast, that is the intercellular spaces or xylem vessels, causing
parenchymatousand vascular or parenchymatousvascular diseases, respectively. Be-
sides the endophytic habitat, some bacterial species also have the capacity to survive as
epiphytes on plant surfaces (phylloplane, rhizoplane, carpoplane, etc.). From an epide-
miological point of view, this poses a particular danger becausethey are quick to infect
plants in favourable conditions. Once inside plant tissues, bacteria may implement two
main attack strategies to exploit the host plant nutrients: biotrophy, in which the plant

14
Soil Microbes and their interaction with plants

cells are kept alive as long as possible and bacteria extract nutrients from live cells,
and necrotrophy, in which bacteria kill plant cells and extract nutrients from dead cells.
Interactions ofbacteria with plants can be either incompatible or compatible. Incom-
patible interactions occur when the bacterium encounters a non-host plant (non-host
resistance) or a resistant host plant (cultivar-specific resistance) and they are frequently
associated with a hypersensitive response (HR), i.e. a rapid, programmed death of plant
cells that occurs at the site of infection. Compatible interactions occur when the bacte-
rium infects susceptible host plants causing disease symptoms.

3.1 Bacterial pathogenicity and virulence

To avoid any confusion in terminology, it is important to define the terms patho-

genicity and virulence, which are often erroneously considered synonyms. According
to Shurtleff and Averre [Scurtleff & Averre et al. 1997], pathogenicity is, ‘the ability of
a pathogen to cause disease’, while, ‘virulence is the degree or measure of pathogenic-
ity of a given pathogen’. Therefore, a bacterium could be pathogenic yet have varying
degrees of virulence. Pathogenicity and/or virulence of Gram-negative plant patho-
genic bacteria is strictly dependent on the presence of secretion apparatuses in their
cells, through which they secrete proteins or nucleoproteins involved in their virulence
in the apoplast or inject in the host cell. To date, five secretion systems, numbered from
I to V have been described in both animal and plant Gram-negative pathogenic bacteria
[Desvaux et al. 2004]. In addition, it is now emerging that the expression of virulence
factors is under the control of quorum sensing, a communication mechanism by which
bacteria regulate the expression of certain genes in response to their population density.

3.2 Pathogenicity and type III secretion system

The pathogenicity of a number of biotrophic Gram-negative bacteria in the gen-

era Pseudomonas, Xanthomonas, Ralstonia, Erwiniaand Pantoeais mainly due to their
ability to produce a type III secretion system (T3SS), also called injectisome [Desvaux
et al. 2004], by which the bacterium injects proteins involved in its virulence into plant
cells (T3SS effectors). It is worth remembering that if the effector leads to the develop-
ment of disease symptoms in the plant it is called a virulence protein, while if it triggers
a defense reaction that leads to the HR, it is referred to as an avirulence protein. The
T3SS is encoded by hrp(HR and pathogenicity) and hrc(HR and conserved) genes,
whose mutations eliminate bacterial pathogenicity in susceptible host plantsand the
ability to elicit HR in non-host or cultivar-specific resistant plants. The hrcgenes are
so called as they encode proteins homologous to the T3SS described in Gram-negative
bacterial pathogens of mammals in the genera Yersinia, Shigellaand Salmonella, which
are thought to be the core components of this secretion apparatus.

15
 plant- pathogen interaction

3.3 Agrobacterium tumefaciens infection process

The particular infection modality of Agrobacterium tumefaciens, the causal

agent of crown gall in manywoody and herbaceous dicotyledonous plants, is worthy of
separate discussion. This Gram-negative soil bacterium is able to genetically transform
healthy host cells in tumoral cells by inserting a part of its DNA (transferred DNA;
T-DNA), contained on the tumor-inducing (Ti) plasmid, into the plant genome [Sheng
et al. 1996].

3.3.1 Other bacterial virulence factors

The severity of disease symptoms caused by bacteria in plants is determined by

the production of a number of virulence factors, including phytotoxins, plant cell-wall-
degrading enzymes, extracellular polysaccharides and phytohormones. In general,
phytopathogenic bacterial strains mutated in any virulence factor more or less reduce
their virulence, while their pathogenicity remains unchanged.

3.4 Phytotoxins

Phytopathogenic bacteria of the Pseudomonasgenus, especially Pseudomonas

syringae, produce a wide spectrum of nonhost-specific phytotoxins, i.e. toxic com-
pounds causing symptoms in many plants independently of the fact that they can or
cannot be infected by the toxinproducing bacterium. On the basis of the symptoms
they induce in plants, phytotoxins of Pseudomonasspp. have been grouped in necrosis-
inducing and chlorosis-inducing phytotoxins. P. [Link], the causal agent
of many diseases and types of symptoms in herbaceous and woody plants, produces ne-
crosis-inducing phytotoxins, lipodepsipeptides, which based on their amino acid chain
lengthare usually divided in two groups: mycins (e.g. syringomycins) and peptins (e.g.
syringopeptins) [Melotto et al. 2006]. Syringomycins and syringopeptins are synthe-
sised by modular nonribosomal peptide synthases, whose chromosomal genes are pres-
ent in the syrand sypclusters, respectively. These clusters are adjacent to one another
inside the syr-sypgenomic island. Both phytotoxins induce necrosis in plant tissues and
form poresin plant plasma membranes, thereby promoting transmembrane ion flux and
cell death [Bender et al. 1999]. Since all strains of P. syringaepv. syringaeanalyzed
produce both syringomycins and syringopeptins, interrelated roles for the toxins in
the plant pathogen interaction have been suggested. The advantages of producing two
phytotoxic cytotoxins during P. syringaepv. syringae pathogenesis are unclear. Studies
carried out to determine their contribution to virulence, based on assays of mutants de-
fective in the synthesis of one or both types of lipopeptide toxins, provided contrasting
results in the function of the host used. Mutations in P. syringaepv. syringaeof either
the syrB1(syringomycin synthetase) or the sypA(syringopeptin synthetase) genes pro-

16
Soil Microbes and their interaction with plants

voked a reduction in virulence of the bacterium in immature cherry fruits, which was
strongest in a syrB1-sypAdouble mutant [Bender et al. 1999]. By contrast, the viru-
lence of a mutant for the syrB operon in P. syringaepv. syringae strain B728a did not
change virulence on bean [Bender et al. 1999].

3.5 Plant defense responses against bacterial attacks

During their lifetime, plants are continually exposed to a vast number of poten-
tial phytopathogenic bacteria, against which they try to defend themselves through a
multilayered system of passive and active defense mechanisms. When defense respons-
es are effective in contrasting them, plants are considered resistant. Most plant spe-
cies are resistant to most species of potential bacterial invaders, a phenomenon termed
non-host or species resistance [Nürnberger et al. 2004]. It is likely that during evolu-
tion, non-host resistance has been overcome by individual phytopathogenic strains of
a given bacterial species through the acquisition of virulence factors, which enabled
them to either evade or suppress plant defense mechanisms [Nürnberger et al. 2004]. In
such cases, plants that became hosts to such bacteria were rendered susceptible to their
colonization and disease ensued (compatible interaction). Furthermore, within a single
host plant, resistance to bacterial diseases can also occur at the level of individual
cultivars and it is termed host resistance and in particular cultivar-specific resistance.
As a result of coevolution between host plant and the host bacterium, individual plant
genotypes have evolved resistance genes that specifically recognize bacterial strain
or racespecific factors and allow the plant to resistto the infection of this particular
race (race-specific resistance or cultivar-specific resistance) [Nürnberger et al. 2004].
In general, cultivarspecific resistance conforms to the gene-for-gene hypothesis and is
genetically determined by complementary pairs of bacterium-encoded avirulence (Avr)
genes and plant resistance (R) genes. Most Avr proteins are considered virulence fac-
tors required for the colonization of host plants, which act as specific elicitors of plant
defense in resistant host cultivars as they trigger the plant surveillance system [Nürn-
berger et al. 2004]. Non-host resistance of the type II and cultivar-specific resistances
to phytopathogenic bacteria, are typically associatedwith HR, which is characterized
by the localised rapid death of host cells at the infection site and contributes to limit the
growth and spread of the invading bacterium. However, there are some plant-bacterium
interactions characterized by non-host resistance not involving the HR (type I non-host
resistance), which mainly rely on the so-called basal resistance or innate immunity.
Similarly to the innate immunity in mammals, basal resistance is elicited in plants by
pathogenassociated molecular patterns (PAMPs) or, more precisely, by microbialasso-
ciated molecular patterns(MAMPs),such as in the case of bacteria, lipopolysaccharide,
flagellin, and cold shock protein. Basal resistance is also induced outside of plant cells
by non-pathogenic bacteria, TTSS mutants and heat-killed bacteria and manifests in the

17
 plant- pathogen interaction

formation of a callose-rich papilla beneath the plant cell wall, localized production of
reactive oxygen species and increased expression of phenylpropanoid pathway genes
and of other defense associated genes.

Figure 4. Some defence mechanisms in plants

3.6 Host-parasite relationships

Host and parasite cells cannot coexist harmoniously. Thus, anincompatible

cellular relationship between the parasite and host is essential fordisease develop-
ment. If the toxins used to kill host cells are not released at theright time, place
or concentration, or if a particular host genotype is insensitive tothe toxin, host
cells will not die. The necrotroph will be unable to colonise orreproduce and the
plant will be resistant. Two types of necrotrophic pathogensexist: (i) those with
a wide host range involving many plant species and (ii) those with a host range
restricted to a few plant species or even to cultivars within aspecies. The key
difference between these two types of necrotroph is thespecificity of the toxin(s)
produced. Necrotrophs with a broad host range secretetoxins that act on metabolic
targets common to many plants. In contrast, thepathogenic ability of necrotrophs
that release host-specific toxins is conditionedby the gene that encodes the ability
to produce the toxin and by a gene insusceptible cultivars of the host that en-
codes sensitivity to that toxin. Hostspecific necrotrophs usually form a pathogenic
race or pathotype structure wheresome races can attack some cultivars within a
species but not others. If the genethat conditions sensitivity to a particular host-
specific toxin is absent from acultivar, that cultivar will be resistant to the disease
caused by that [Link] on the other hand are obligate parasites that
obtain nutrients fromliving cells. Consequently, they must establish a compatible
cellular relationshipwith their hosts. Biotrophs act as ‘sneaks’. They typically in-
fect through naturalopenings or by directly penetrating their host’s surface. They
mostly then growbetween the cells of their host and only penetrate host cell walls
(but not host cellmembranes) to form food-absorbing haustoria. The pathogen

18
Soil Microbes and their interaction with plants

develops withouteliciting the host’s defence responses or by spreading in advance

of the plant’sability to activate its defence responses. The level of specialisation
required toestablish this type of relationship usually means that biotrophs have a
restrictedhost range and a well-defined pathogenic race structure. If host cells die
inadvance of invasion by a biotrophic pathogen, the plant will be resistant becaus-
ethe pathogen is unable to establish a parasitic relationship. A second factor that
influences whether a parasitic relationship will becomeestablished is the way that
the plant under challenge responds. Someinteractions between individual pathogen
propagules and plant cells may lead tosuccessful pathogen establishment, while
others may not. In this chapter it willbecome evident that resistance or suscep-
tibility of a whole plant and plantcommunities is the sum of many individual
cellular interactions. Plants that areresistant restrict or retard the development and
reproduction of an overwhelmingmajority of individual pathogen propagules that
attack it. In this sense resistanceis quantitative-resistant hosts prevent or slow the
development andreproduction of a higher proportion of pathogen propagules than
susceptiblehosts. For the purposes of plant breeding, the response of a plant to
pathogeninoculation is often categorised as either’resistant’or’susceptible’, although
froma cellular perspective this distinction is not always so clear. Resistance and-
susceptibility are more accurately portrayed as the extremes of a continuumupon
which most host-parasite interactions sit. Resistance may be expressed inmany
ways, from the inhibition of propagule germination and penetration, thekill-
ing of pathogens before establishment, to the restriction or retardation ofcolony
development and reproduction once the pathogen has established. Forexample,
different genes for stem rust resistance in wheat act at different stagesof the
host-parasite interaction. Some cause the rapid death of the pathogenfollowing
attempted penetration, others allow initial infection, but preventhaustorial develop-
ment and starve the pathogen, while the ‘slow-rusting’ genesallow parasitism and
pathogen reproduction, but at a much slower rate than insusceptible cultivars.
Each type of interaction provides useful resistance for plantbreeders because they
all delay the onset of epidemics and reduce yield [Link] early steps involved
in the establishment of a host-pathogen relationshipare delicate and sensitive to
environmental factors, including the presence ofother micro-organisms. The host-
parasite-environment interaction is mediated bya complex interchange of signals.
Plants respond to pathogen attack by erecting ahighly coordinated series of mo-
lecular, cellular and tissue-based defence barriers. All plants have the capacity to
activate these defences. However if they areactivated too little, too late, or in the
wrong place, they will fail to restrict thepathogen and the plant will be suscep-
tible. Pathogens respond by escaping orsuppressing plant defence responses or by
rendering these responses impotent,for example by detoxiffing plant antibiotics.
The interaction of pathogen nutrient requirements and host responses leads tofive
possible outcomes if environmental conditions favour infection (Fig. 5)

19
 plant- pathogen interaction

No relationship is established when the plant and the pathogen ignore

eachother. For example, a spore of a fungus may germinate, but because the host-
does not provide essential requirements for pathogen development, theresulting
hypha fails to penetrate or establish a parasitic relationship. Thefungus dies when
its energy reserves are exhausted. The plant does not reactin any way and is re-
sistant by default. It is a non-host.

Figiure 5. Five possible relationships between plants and potential pathogens.

4. Rhizoremediation studies
One of the classical papers in the field of ‘natural’ rhizoremediation is a report
by Radwan and colleagues (1995) showing that plants growing in sand contaminated
by oil spills after the Gulf War exhibited clean roots due to the removal of aromatic
hydrocarbons by microorganisms. Although there are many reports about rhizoreme-
diationexperiments under laboratory conditions, there are fewer examples in thescien-
tific literature detailing the successful removal of pollutants from contaminated soils in
‘real’ scenarios via theconcept of ‘designed’ rhizoremediation. so we will only present
a few examples thathave not been included in these reports and in whichdifferent ex-
perimental approaches have been [Link] is a highly persistent
pollutant; itsg-isomer (lindane) has frequently been used aspesticide and is widespread
throughout the biosphere.AlthoughSphingomonasUT26 is a well-characterizedlin-
dane-degrading bacterium, it was not able to proliferate in soil, probably due to its high
sensitivity to the lowwater content in the soil. Böltner and colleagues (2008) set up a
double enrichment approach in which both lindane degradation and root proliferation
of bacteria were prerequisites for [Link] yieldedSphingomonasstrains that were
now ableto proliferate in the plant root. Greenhouse assaysrevealed that up to 30% of
lindane in soil can beremoved in a 3-month [Link] the case of TNT removal, over
90% of TNT in the soilcould be removed in field experiments through the combined
action of phytoremediation and rhizoremediation. Inthese assays, phytoremediation
proved more efficientthan rhizoremediation, but bacteria played a key role inthe es-
tablishment of plants at the polluted [Link] several PCB degraders have been

20
Soil Microbes and their interaction with plants

identified, PCB degradation is [Link] of the best inducers of PCB degradation

is biphenyl, which obviously cannot be used as a soil amendment to promote PCB
elimination. Several attempts topromote PCBs rhizoremediation via the introduction
ofthe bphgenes under the control of different promoters inP. fluorescensF113 (a strain
with good colonization abilities) have had limited success. In a different approach,
Narasimhanand colleagues (2003) used the ability ofP. Putida PML2to degrades phen-
ylpropanoid compounds to promotePCB degradation. InArabidopsis thaliana, 37% of
rootexudates were flavonoids and most of these were phenylpropanoids. They showed
that a wild-type strainP.putidaPML2 was able to establish in the rhizosphere ofArabi-
dopsisplants better that an auxotrophic mutantthat was unable to use phenylpropanoids
for [Link] both mutant and wild-type strains presentedsimilar growth on
different PCBs in liquid cultures, PCBelimination was higher when the parental strain
wasintroduced in gnotobiotic systems with Arabidopsis, thanwhen the mutant was used.
Polychlorinated biphenylelimination was inferior if anArabidopsismutant unableto
produce phenylpropanoids was used. These experiments showed that rhizoengineer-
ing, the modification ofmicrobial populations in the plant roots for biotechnological
purposes, is a valuable option to enhance contaminant [Link] pro-
teins to be expressed at the surface ofthe microbial cell is another promising strategy
for pollutant removal, especially for the removal of heavy [Link] strategy has
recently been reviewed by Saleem andcolleagues (2008) and we refer the reader to this
reviewfor more information.
4.1 Beneficial effect of the rhizosphere microbial

Figure 6. Ecological factors influencing the root exudation process and thereby rhizo-
sphere colonization by beneficial rhizobacteria

21
 plant- pathogen interaction

4.2 Rhizosphere colonization

Microbial attachment to and proliferation on roots is generally referred to as

root colonization. Root colonization is an important factor in plant pathogenesis of soil-
borne microorganisms as well as in beneficial interactions used for microbiological
control, biofertilisation, phytostimulation, and phytoremediation (Thomas et al., 2008).
The beneficial effect of PGPR on plant growthvia an enhancement of the nutrient status
can vary depending on where and how the PGPR colonizes the host plant. Since the
1980’s, many studies have focused on the root colonization. PGPR may colonize the
rhizosphere, the surface of the root, or even superficial intercellular spaces (McCully,
2001).Howie et al. (1987) hypothesized a two phase process (Howie et al., 1987). In
phase I, bacteria on the seed attach to the emerging root tip and are passively trans-
ported into the soil. During root growth some cells remain associated with the tip while
others are left behind on older portions of the root and in the rhizosphere. In phase
II bacteria deposited along the root multiply and form microcolonies in nutrient-rich
microsites, compete with indigenous microflora and avoid displacement. The concept
of phase I and II is meant to define more easily two different stages, rather than a strict
temporal sequence of events because both phases occur simultaneously on different
parts of the roots (Weller and Thomashow, 1994). Some authors reported that bacterial
cells first colonize the rhizosphere following soil inoculation (Gamalero et al., 2009).
Then, bacterial cells have been visualized as single cells attached to the rootsurfaces,
and subsequently as doublets on the rhizodermis, forming a string of bacteria (Com-
pant et al., 2010). Colonization may then occur on the whole surface and bacteria can
even establish as microcolonies or biofilms (Benizri et al., 2001). Nevertheless it is
important to note that the root system is not colonized in a uniform manner, different
population densities being reported for the diverse root zones. For example, Kluyvera
ascorbata colonized the upper two-thirds of the surface of canola roots, but no bacte-
ria were detected around root tips (Vessey, 2003). Non-uniform bacterial colonization
along the root can be explained by different factors such as varying root exudation pat-
terns, bacterial quorum sensing effects as well as many others (Compant, et al., 2010).
Root colonization is influenced by many factors: biotic, such as genetic traits of the
host plant and the colonizing organism or abiotic such as growth substrate, soil hu-
midity, soil and rhizosphere pH, and temperature. This is why notany soil bacteria can
colonize root system. In order to colonize the rhizosphere during an extended period
characterized by strong microbial competition and exert plant growth promoting traits,
soil bacteria need to be rhizosphere competent (Whipps, 2001). Root colonization and
rhizosphere competence are heavily influenced by the plant changes in the physical and

22
Soil Microbes and their interaction with plants

chemical composition of the rhizosphere soil compared to the bulk soil. These differ-
ences are manifested by changes in water potential, partial pressure of O2, and other
physical and chemical characteristics due to plant exudations (Vessey, 2003).

There are several reports that link rhizosphere colonization to root exudation.
Root exudates (carbohydrates, amino acids, organic acids) and mucilage-derived nutri-
ents attract deleterious rhizobacteria as well as beneficial and neutral bacteria allowing
them to colonize and multiply inthe rhizosphere (Walker et al., 2003). Consequently
PGPR have to be highly competitive to successfully colonize the root zone (Compant,
et al., 2010).

4.3 Mechanisms of plant growth promotion

Investigations of the plant growth promotion mechanisms by PGPR are increas-

ing at a rapid rate in order to use the best PGPR strain as commercial biofertilizer
(Bhattacharyya and Jha, 2012). Many reports present, from different perspectives, the
PGPR activities involved in plant growth enhancement. Further we present the most
cited mechanisms used by PGPR to promote plant growth. As colonization occurs,
PGPR interact with the plant host and enhance the nutrient uptake of host plants by (1)
biological N2fixation, (2) increasing the availability of nutrients in the rhizosphere, (3)
inducing increases in rootsurface area, (4) enhancing other beneficial symbioses of the
host, and (5) combination of modes of action (Vessey, 2003).

4.4 Biological N2fixation

Biological N2fixation (BNF) is restricted to prokaryotic organisms. The most

studied and longest exploited PGPR are the rhizobia (including Allorhizobium, Azorhi-
zobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium) for their
ability to fix N2in their legume hosts (Vessey, 2003). As these microorganisms may
be autotrophic or heterotrophic, different strategies for garnering energy (particularly
carbohydrates) have evolved in symbiotic rhizospheric-associations with plants as well
as in free-living nitrogen-fixing organisms (Cocking, 2003). On average, symbiosis
have the highest nitrogen fixation capability not only because energy (in the form of
carbohydrates) is provided by the plant, but also other conditions (e.g., export of re-
duced nitrogen) are optimized for efficient nitrogen fixation (Cocking, 2003). Symbi-
otic nitrogen fixation by rhizobia in legumes has a profound impact upon agriculture
and human endeavour. This symbiosis involves intimate mutual interaction between
both partners, with altered morphology of both the bacteria and the plant (Mukerji,
et al., 2006). Because of the practical and theoretical importance of the symbiotic ni-

23
 plant- pathogen interaction

trogen fixation there are many review articles and monographs that cover this topic
(Hansen, 1994, Rascio and La Rocca, 2008, Salvagiotti et al., 2008). Also even many
PGPR present the ability to fix N2, yet rarely is their mode of action for the stimulation
of plant growth credited to [Link] is little evidence that PGPR with the ability to
fix N2stimulate the growth of a specific host plant using nitrogenase activity (Vessey,
2003). This is why BNF is not reviewed here, our focus being on most frequent PGPR
mechanisms of action.

5. Mechanisms underlying beneficial plant – fungus interactions in mycorrhizal

symbiosis

Owing to their filamentous organization, fungi exploit very diverse substrates

on the basis of their nutritional strategy. Saprobes thrive in soil, water and on decaying
animal and plant tissues. A smaller group of fungi, the parasitic and mutualistic symbi-
onts, feed on living organisms [Carlile et al. 2001]. Such a classifi cation cannot easily
be applied to mycorrhizal fungi, a heterogeneous group of species spread over diverse
fungal taxa. Although they can spend part of their life cycle as free-living organisms,
mycorrhizal fungi always associate with the roots of higher plants, indeed over 90 % of
plant species, including forest trees, wild grasses and many crops. Both partners benefit
from the relationship: mycorrhizal fungi improve the nutrient status of their host plants,
influencing mineral nutrition, water absorption, growth and disease resistance, whereas
in exchange, the host plant is necessary for fungal growth and reproduction [Smith
& Read, 2008]. Mycorrhizal fungi colonize environments such as alpine and boreal
zones, tropical forests, grasslands and croplands. They have a major role in nutrient
cycling through the specific activity of their mycelium in absorbing soil nutrients and
supplying them to the plant, although their role in carbon flux is less well defined [Sel-
osse & Roy, 2009]. Th e term mycorrhiza is derived from the Greek words for ‘ fungus
’ and ‘ root ’ . Mycorrhizal fungi develop an extensive hyphal network in the soil, the
aptly named wood-wide web [Helgason et al. 1998], which can connect whole plant
communities offering effi cient horizontal transfer of nutrients. Mycorrhizas develop
specialized areas, called symbiotic interfaces, to interact with the host plant [Bonfante,
2001]. Mycorrhizal fungi can be divided into two major groups: aseptate endophytes
such as Glomeromycota, or septate Asco- and Basidiomycota. More commonly, my-
corrhiza classifi cations reflect anatomical aspects and identify two broad categories,
referred to as ectomycorrhizas (EMs) and endomycorrhizas, depending on whether the
fungus colonizes the root intercellular spaces or develops inside cells ( Fig. 7 ).

24
Soil Microbes and their interaction with plants

Figure 7 . Illustration of root colonization structures in ectomycorrhizal (blue)

and arbuscular mycorrhizal (pink) interactions.

Endomycorrhizas are further divided into orchid, ericoid and arbuscular mycor-
rhizas (AMs). A descriptive approach has dominated the investigation of mycorrhizas
for at least 50 years until the advent of molecular biology, and the ‘ omics ’ era provid-
ed insight into their mechanisms. Highthroughput technology, genome sequencing of
fungi, plants and associated microbes, transcriptomic analyses, availability of mutant
collections, RNA interference lines and plants transformed with fluorescent tags have
all led to new perspectives on plant – microbe interactions, and on EMs and AMs in
particular. In this review we focus on the mechanisms that govern the growth of EM
and AM fungi and their interactions with plants.

5.1 EM fungi dominate the world forests

Trees in the families Pinaceae, Fagaceae, Dipterocarpaceae and Caesalpinoida-

ceae found in many forests all interact with hundreds of EM species of Basidiomycetes
and Ascomycetes, so that these fungi can be said to have shaped the present forests.
EM fungi colonize the lateral roots of these trees with sheathing mycorrhizas, in which
a fungal mantle covers the root tip, and a so-called Hartig net of intercellular hyphae
surround epidermal and outer cortical cells ( Figs 7). EM fungi may also live inde-
pendently of plant roots, as demonstrated by their growth capabilities in Petri dishes.
Molecular techniques have identified EM fungi in the field, and owing to metagenom-
ics, that is, the deep sequencing of organisms living in an environment, a multitude
of sequences from soil are catalogued, also highlighting the rhizosphere around EMs
as a special biome. Use of the Roche 454 Titanium genome sequencer has revealed
unexpected levels of fungal biodiversity when applied to the soil of a French forest,
25
 plant- pathogen interaction

in which Agaricomycetes were found to be the dominant class including a large num-
ber of EM species. These data confirm that EM fungi have a dual lifestyle: they live
as symbionts with plant roots and as facultative saprotrophs in soil [Martin & Nehls,
2009]. However, data on their in situactivity (metatranscriptomics) are not yet avail-
able and the ecological functions accomplished by EM fungi in the soil remain at the
moment unexplored.

5.2 Deciphering the genome of EM fungi

Genome sequencing of two EM fungi, the Basidiomycete Laccaria bicolor and

the Ascomycete Tuber melanosporum (black truffle), has shed some light on EM biol-
ogy, highlighting similarities and differences between the two fungal symbionts and
identifying the primary factors that regulate mycorrhiza development and function.
Although L. bicolorhas a 65-Mb genome and an estimated,102 protein-encoding genes,
T. melanosporum, with its 125-Mb genome and an estimated 7500 protein-encoding
genes, is in the odd condition of being the largest sequenced fungal genome, with a rel-
atively small expected proteome compared with other filamentous fungi. This argues
that the symbiotic status can be achieved even with a reduced number of protein-cod-
ing genes, very few of which (1.5% ) are differentially regulated during mycorrhizal
formation. Another contrasting feature is that the genome of L. bicolorhas undergone
extensive expansion of a few gene families with predicted roles in protein – protein
interactions and signal-transduction mechanisms, whereas this trait has not been ob-
served in T. melanosporum, in which multigene families are rare. Th e genome size
of both EM fungi is not ascribed to large-scale duplication events, but rather to a high
number of transposable elements that represent more than 20 and 58 % of the genome
in L. bicolor and T. melanosporum, respectively. In the truffle, the insertion of trans-
posable elements has been dated to 2 – 5 million years ago, and is considered to have
contributed to genome evolution and plasticity.

Nutrient and signal exchange in EMs. Mycorrhizal fungi, similar to other

plant-interacting fungi, base their life cycle on the uptake of organic carbon from a
living plant (Fig. 8). Fifteen genes encoding putative hexose transporters have been
annotated in the L. bicolorgenome and many of them are upregulated during symbiosis.
However, the genome lacks genes encoding invertases, the enzymes that hydrolyse su-
crose (the most abundant sugar in plant tissues) to glucose and fructose, suggesting that
L. bicolordepends on the glucose released by its green host. In contrast, T. melanospo-
rum possesses one invertase gene, indicating its potential capacity to access the plant
sucrose pool, whereas other EM fungi, Amanita muscariaand Hebeloma cylindrospo-
rum, also metabolize fructose. Interestingly, Ustilago maydis, a biotrophic pathogen
causing smut disease on maize, possesses a sucrose transporter localized on the plasma
membrane [Wahl et al, 2010] that allows the fungus to directly acquire sucrose with no
need for secreted invertases. Such a range of mechanisms to access plant sugars thus
26
Soil Microbes and their interaction with plants

seems to mirror some of the differences in nutritional strategies between symbiotic and
pathogenic fungi. Mutualistic interactions are based on the exchange of nutrients; for
this reason, genes coding for transporter proteins are expected to be well represented
in EM fungal genomes.

Figure 8. Scheme summarizing the main nutrient exchange processes in EM and AM

symbiosis

In spite of this prevision, the number of transporters annotated in L. bicoloris

comparable to that of other saprotrophic and pathogenic fungi (491 genes), and they
are even less abundant in T. melanosporum (381 sequences). However, many of these
genes are upregulated in the symbiotic condition, implying very intense traffic of ami-
no acids, oligopeptides and polyamines through the symbiotic interface. Analysis of
EM genomes highlights the capacity of the freeliving mycelium to import organic and
inorganic N sources, including nitrate, ammonium and peptides from the soil, through
proteins such as the ammonium transporter or the urea permease (Fig.3). These func-
tions are probably common to many EM fungi, as they were fi rst described in A. mus-
caria (AmAMT2) and Paxillus involutus (PiDur3). From the mycelium, N compounds
reach the mantle and the Hartig net, from where they can be exported to the plant; even
if this mechanism is poorly understood, glutamine and ammonium are the best candi-

27
 plant- pathogen interaction

date molecules to cross the symbiotic interface. Th is implies ammonium assimilation

into amino acids and their translocation to the plant, or, alternatively, the direct release
of ammonium ions from the Hartig net hyphae. A possible confirmation of the second
alternative comes from the upregulation of ammonium importers in EM poplar roots.
Pathogenic fungi possess genes for a large array of secreted /effector proteins. Th e
discovery that L. bicolorpossesses about 300 small cystein-rich secreted proteins that
are upregulated during interaction with poplar and Douglas-fir opens new scenarios.
For example, these proteins could function as effectors during the early phase of the
plant – fungus interaction or could be involved in the construction of the symbiotic in-
terface, as suggested by the immunodetection of one of these proteins (MISSP7) in the
hyphal mantle. These data point to the interface area as a site of recognition between
the partners; however, comparable effector-like proteins have not been detected in T.
melanosporum, leading to the question of whether EM Ascomycetes have developed
alternative pathways for communicating with their hosts or whether such functions are
carried out by other small secreted proteins.

5.3 Finding the host root

The dissection of plant responses illustrates how the mechanisms operating to

accommodate the AM fungus inside the plant cell lumen are shared by diverse plants
and have been conserved during evolution. Root colonization is vital to AM fungi (Fig.
9). Th eir spores feed germinating hyphae through the catabolism of storage lipids
for just a few days. During this period, hyphae explore the soil in search of a host but
if they never meet one, they arrest their growth and retract their cytoplasm back into
the spore, which may again become dormant and restart the germination process over
and over. This situation is not likely to be very frequent in nature because of the wide
host range of these fungi, but is a hallmark of their obligate biotrophy, as well as their
endurance. Th is strict biotrophy has long frustrated attempts to obtain enough mate-
rial to perform large-scale nucleic acid extractions or produce sterile inoculum. The
latter was made possible by root organ cultures that serve as a living substrate to grow
several species of AM fungi [Bécard, G. & Fortin, 1988]. Strigolactones as a new class
of bioactive molecules. Perception of the host plant by the presymbiotic mycelium is
mediated by root exudates. The responsible compounds that are released by the root
and diffuse a short distance, before being washed away or degraded have been identi-
fied as strigolactone. They stimulate AM fungal meta bolism and branching, a change
in mycelial growth pattern that is thought to increase the chances of an encounter with
the host.

28
Soil Microbes and their interaction with plants

Figure 9. Schematic summary of the root colonization process by AM Fungi

Strigo lactones also have a hormonal role in inhibiting lateral branching of

shoots. Th is direct effect on plant development is considered to be the main function
for this class of molecules, which are in fact produced by many plant taxa, including
those not associated with mycorrhizal fungi. Th e leakage of strigolactones from roots
into the soil and their rapid hydrolysis have in any case made them ideal for signalling
root proximity in the rhizosphere. The seeds of parasitic weeds are also stimulated to
germinate by strigolactones [Matusova et al. 2005]. These multiple functions recall the
pleiotropic effects of auxins, and promote consideration of strigolactones as hormones.

6. Plant-virus interaction

The molecular analysis of plant-virus interactions, and the evolution of plant

virus populations. The link between these two research lines is the understanding of
resistance of plants to viruses and of the processes in virus populations that result in the
overcoming of resistance factors.

29
 plant- pathogen interaction

Figure 10. Collapse of tomato plants in a greenhouse crop affected by a viral

disease.

6.1 Molecular analysis of plant-virus interactions.

Viruses are important pathogens of crops for which the only direct control strat-
egy is the use of resistance cultivars. A common mechanisms of resistance is interfer-
ence with virus colonisation of the plant host. One major interest of the group is the
analysis of virus- and host-plant-encoded factors that determine the systemic infection
of a plant by a virus. We have analysed, for different viruses, the structural forms in
which virus infection translocates within the phloem and/or the xylem, and the role of
capsid proteins as determinants of systemic movement. Present emphasis is on the iden-
tification and characterisation of phloem proteins that interact with the viral structures
in sieve elements, and on the role of these proteins in systemic movement. A second
interest is on plant reactions to infection that results in a decreased virus accumula-
tion (quantitative resistance) or on a lesser effect of infection on symptom develop-
ment and productivity decrease (tolerance). We are interested in the characterisation
of mechanisms of quantitative resistance and of tolerance, including the identification
and analysis of QTLs involved in these traits. Last, we are interested in the genera-
tion of defective RNAs (D-RNAs) and on the role of D-RNAs and satellite RNAs as
modulators of viral pathogenicity. D- RNAs will also be used as models to analyse
viral functions such as encapsidation, replication, transcription or movement.

30
Soil Microbes and their interaction with plants

6.2 Evolution of plant virus populations

Most used resistance factors have been eventually overcome by the appearance
of resistance-breaking strains, due to the evolution of pathogen populations under the
selective pressure of the resistance. The goal of this research line is to understand
the evolutionary factors that model the genetic structure plant virus populations and
that may result in the evolution of virulence. This is approached by the analysis of the
genetic structure of virus populations in nature and by evolution experiments under
controlled conditions. Research on virus evolution is organised along three axes. The
first is the analysis of the adaptability of plant viruses, and how easily they respond to
selection: High mutation rates in RNA viruses may not be adaptive, as indicated by
the large fraction of deleterious and lethal mutations. Also, the high rates of genetic
exchange by recombination or reassortment of genomic segments may be coun-
tered by selection against heterologous gene combinations due to epistatic interactions
among viral genes. The effects of selection could also be hindered by random genetic
drift, and we are interested in the quantitative analyses of population bottlenecks
along the virus life cycle, which might significantly reduce the effective population
size. The second axis is centred on the evolution of virulence, and its relationship with
different epidemiological factors such as within host multiplication and between
host transmission rates, or host range and the trade-offs associated to the adaptation
to different host species or genotypes. The third axis considers the epidemiology
of viruses, including their interactions with other viruses in mixed infections, and
the effects of host and vector range on the virus population dynamics. Hence, our long
term goal is to reach a global understanding of the population biology of plant viruses.
Recently, a new research line has been started, which will address some of the aspects
detailed above for phytopathogenic fungi.

6.3 Genetic exchange information

Genetic exchange through recombination and reassortment of genomic seg-

ments (in viruses with a segmented genome) is an important generator of genetic
variability with a potentially high impact on virus evolution: genetic exchange often
results in large genotypic effects and may be at the root of hosts switches, host range
expansions and virus emergency. We have analysed the role of genetic exchange in
shaping the genetic structure of the populations of three different RNA viruses: Water-
melon mosaic virus (WMV), Pepino mosaic virus (PepMV) and CMV. Recombinants
among different genotypes, and reassortants for CMV, were found in field populations
of all three viruses, however, selection against recombinants with hybrid proteins, and
against reassortants seems to be quite effective (Moreno et al. 2004; Bonnet et al. 2005;
Pagán et al. 2006), a result consistent with the hypothesis of co-adaptation of genes
within viral genomes. A prerequisite for recombination is the occurrence of mixed in-
fections, which were detected for all three viruses. More interesting is the observation

31
 plant- pathogen interaction

that positive association of different viruses in mixed infections seems to be the rule
for plants viruses, as shown in an analysis of the prevalence of five virus species in
twenty one wild host plant species (Malpica et al. 2006). Host adaptation and the pos-
sible trade-offs involved was analysed for CMV. CMV is a typical generalist pathogen,
with a large host range. The population dynamics of CMV in different hosts, including
crops, was analysed. CMV prevalence was dependent of plant density, and population
dynamics was uncoupled in wild hosts and crops (Sacristan et al. 2004). However, the
genetic structure of the CMV population did not suggest adaptation of different CMV
genotypes to different host species (Bonnet et al. 2005). Host adaptation was further
analysed in a serial passage experiment involving three host plant species and six
CMV strains. Differential accumulation of the CMV strains in the different hosts did
not provide evidence for host adaptation, but host associated differential infectivity
did. Moreover, data suggested that trade-offs related to host adaptation could occur
(Sacristán et al. 2005). It could be that host adaptation, and associated trade-offs may
be too subtle to be detected in a host passage experiment, but could be uncovered by
the analysis of natural populations. This was indeed the case: to analyse data on the
prevalence of five generalist viruses in 21 wild plant species, indices to measure host
and virus associations, and their statistical significance, were developed. Analyses
showed that host selectivity was the rule for this viruses and, more important, that the
more prevalent viruses were the most host-selective (Malpica et al. 2006). This sug-
gests that host selectivity is a successful strategy with generalist plant viruses shows in
(fig. 11).

Fig 11. Effect of different CMV isolates on Arabidopsis plants

7. Applications

Plants are involved in a complex network of interactions with microorganisms;

32
Soil Microbes and their interaction with plants

some of those are beneficial, others are detrimental, but the former are by far the largest
and still widely unexplored part. There is a growing worldwide demand for sound and
ecologically compatible environmentally friendly techniques in agriculture, capable of
providing adequate nourishment for the increasing human populationand of improving
the quality and quantity of certain agricultural products. For these reasons, the applica-
tion of beneficial microorganisms is an important alternative to some of the traditional
agriculturaltechniques which, as it has been well documented, very often severely alter
the agroecosystem balance and cause serious damage to health. For example, con-
tamination of groundwater by leaching of nitrogen fertilizers, accumulation of nitrates
and persistence of chemicals used in crop protection in edible portion of foods are
cause of grave concern. The use of beneficial microorganisms in the replacement or the
reduction of chemicals has been so far attested (Dobbelaere et al., 2003; Burdman et
al., 2000). Beneficial microorganisms such as diazotrophs bacteria, biological control
agents (BCAs), plant growth promoting rhizobacteria (PGPRs) and fungi (PGPFs), can
play a key role in this major challenge, as they fulfil important ecosystem functions for
plants and soil (Whipps, 1997; Raaijmakers et al., 2009; Hermosa et al., 2011). More-
over, modern agriculture, based on the cultivation of a very limited number of crop spe-
cies and cultivars, is susceptible to epidemic diseases traditionally contrasted through
the use of chemicals. With most crops, no effective fungicides are available against a
lot of fungal diseases. Plant growth stimulation and crop protection may be improved-
by the direct application of a number of microorganisms known to act as bio-fertilizers
and/or bio-protectors. How beneficial microorganisms really do act to improve plant
rooting is only partially known, as several aspects have to be considered, including (i)
the production of metabolites related to root development growth and pathogen control
(phytohormones, antimicrobials, antibiotics), and (ii) the difficulty to discriminate the
direct effects on the specific/total activities and the indirect effects due to the enhanced
availability of nutrients and growth regulators.

7.1 Ecological implications

Soil-borne microorganisms interact with plant roots and soil constituents at the
root-soil interface, where root exudates and decayingplant material provide sources
of carbon compounds for the heterotrophic biota (Barea et al., 2005; Bisseling et al.,
2009). The number of bacteria in the rhizosphere (thenarrow region of soil that is di-
rectly influenced by root secretions and associated soil microorganisms) and rhizo-
plane (the external surface of roots together with closely adhering soil particles and
debris) is higher than in the soil devoid of plants; this happens because soilsdevoid
of plants are poor in many attractive substances secreted from the roots. As soonas a
seed starts to germinate, a relatively large amount of carbon and nitrogen compounds
i.e., sugars, organic acid, aminoacids, and vitamins are excreted into the surround-
ing environment. This attracts a large population of microorganisms inducing vigor-
ous competition between the different species (Okon, 1994). Moreover, rhizosphere
33
 plant- pathogen interaction

microbiomes typically differ between plant species (Bisseling et al., 2009). Beneficial
microorganisms are known to be biocontrol agents and/orgrowth promoters. There are
several modes of action by which theycan be beneficial to plant health, which can be
related to an indirect or a direct positive effect. Microorganisms have indirect positive
effects on plants, affecting adversely the population density, dynamics and metabolic
activities of soil-borne pathogens, mainly through competition, antibiosis, lysis, and
hyperparasitism. Competition takes place for space and nutrients at the root surface;
competitive colonization of the rhizosphere and successful establishment in the root
zone is a prerequisite for effective biocontrol. Antagonistic microorganisms can often
produce a range of different antimicrobial secondary metabolites, and/or extracellular
lytic enzymes. Hyperparasitism is well documented for Trichoderma; it involves secre-
tion of chitinases and cellulases, contact with the pathogen, coiling ofhyphae around
the hyphae of the pathogen, enzymatic digestion of its cell wall, and penetration. Di-
rect positive effects on plants are exerted by rhizosphere microorganisms through a
phytostimulation and a biofertilization of plants; these processes involve production
of phytohormones, non-symbiotic nitrogen fixation, and the increase of availability of-
phosphate and other nutrients in the soil (Burdman et al., 2000). Numerous compounds
thatare toxic to pathogens, such as HCN, phenazines, pyrrolnitrin, and pyoluteorin as
well as, enzymes,antibiotics, metabolites and phytohormones are the means by which
PGPRs act; similarly other phenomena such as quorum sensing and chemotaxis, are
vital for rhizosphere colonization (Castro-Sowinski et al., 2007; Ramette et al., 2011;
Jousset et al., 2011).

7.2 Tolerance to salinity

Soil salinity in arid regions is frequently an important limiting factor for cul-
tivating agricultural crops. PGPR-elicited plant tolerance against salt stress has been
intensively studied, showing that inoculation with endophytic bacteria can mitigate the
effects of salt stress in different plant species. High K+/Na+ratios were found in salt-
stressed maize in which selectivity for Na+, K+and Ca2+was altered upon inoculation
with Azospirillum (Hamdia et al., 2004). Similarly, inoculation of pepper with Bacillus
sp.TW4 led to relief from osmotic stress, which is often manifested as salinity (and/or
drought) stress. In these plants, genes linked with ethylene metabolism under abiotic
stress were down-regulated (Sziderics et al., 2007). Because Bacillus sp. TW4 showed
ACC deaminase activity, the authors speculated that the enzyme may be involved in
the lower expressionof these genes. Salt stress has also been shown to affect nodulation
during Phaseolus–Rhizobium interaction. However, secondary inoculation of the salt-
stressed plants with Azospirillu caused an extended exudation of plant flavonoids com-
pared to Rhizobium alone, implying an induction of flavonoid genes in the presence of
Azospirillum (Dardanelli et al., 2008). Thus, the co-inoculation of plants with different
bacterial species may contribute to relieving abiotic stress. IST to salt stress was also

34
Soil Microbes and their interaction with plants

noted with Arabidopsis (Zhang et al., 2008) using Bacillus subtilisGB03, a species
that has previously been used as a commercial biological control agent. Interestingly,
some of the volatile organic compounds (VOCs) that are emitted from B. subtilisGB03
(Ryu et al., 2004) are bacterial determinants involved in IST. The response to saline
stress has also been evidenced in barley seedlings where inoculation with Azospiril-
lumseemed to mitigate NaCl stress (Zawoznik et al., 2011).

7.3 Drought tolerance

Land surface becoming arid or semi-arid has been rising progressively in these
last decades; water use efficiency is a current priority for the United Nations policy
and a key issue for plant research. Under water stress conditions, leaf transpiration and
leaf conductance decrease, and the water use efficiency rises; this mechanism keeps
plant growth under water-limited environments (Aroca & Ruiz-Lozano, 2009). Plant
responses to drought include an increase in abscisic acid (ABA) levels,that cause sto-
matal closure to minimize water loss; these events involve production of activated
oxygen species (Cho et al., 2008). Other plant-signalling compounds are involved in
regulating stomatal closure, such as methyljasmonate, salicylic acid and ethylene. The
mechanisms that allow plants to cope with drought stress are regulated by changes in
gene expression; drought regulated genes can be divided in two groups: functional
genes (encoding for transporters, detoxification enzymes, osmolyte biosynthesis en-
zymes etc.) and regulatory genes, that encode for transcription factors (Aroca& Ruiz-
Lozano, 2009). On the whole, the beneficial effects of PGPR on plant drought tolerance
is caused by changes in hormonal contents, mainly that ofABA, ethylene and cytoki-
nins.

7.4 Experimental considerations about plant-beneficial bacteria

In the Mediterranean area the use of microorganisms became indeed wide-

spread in the ’80s, in coincidence with the sudden spread of soybean crop, that required
the inoculation of the nitrogen fixing Bradyrhizobium japonicum, mainly applied to
seeds as peat based or liquid inocula at sowing. Operators became familiar with the use
microorganisms in agriculture. After that, despite the huge potentiality of beneficial
microorganisms, a relative low diffusion must be highlighted, owing to “inconsistent”
results in field experiments, but also owing to prejudices derived from the easy and
large availability of chemicals. At the moment, as a consequence of (i) a growing in-
terest towards low input agriculture systems (organic farming, biodinamics, natural
farming), (ii) a favourable opinion of consumers for food with no chemicals, and (iii)
the increased difficulties in the employment of chemicals according to the most recent
laws, we are assisting at a “microbiological revolution”, and the use of microorganisms
is increasing.

35
 plant- pathogen interaction

The use of beneficial microorganims is mostly oriented to improve plant growth

and protection in an agricultural context, nevertheless several applications in a wider
environmental sense could be prospected, as reported by our group in scientific litera-
ture. Pseudomonas fluorescent (Russo et al., 1996; 2001; 2005), Bacillus subtilis(Felici
et al., 2008), Rhizobiumspp (Toffanin et al., 2000; Casella et al., 2006), are some of
beneficial bacteria applied in our experimental/scientific work as biofertilizers and/
or biocontrol agents in agriculture. Other potential applications currently include mi-
cropropagation, bioremediationand phytoremediation, phosphate solubilization, soil
aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbingand biogas
production.

7.5 Micropropagation

Micropropagation is an efficient method ofpropagating large numbers of ge-

netically uniform plants (Honda & Kobayashi, 2004), although serious problems con-
cerning specific steps including explant sterilization, media manipulation, and ac-
climatization phase, can often invalidate its success, making the plantlets production
a cost-intensive process. In vitrobacterization of potato plantlets has been shown to
enhance their transplant stress tolerance thereby eliminating the need of anexpensive
greenhouse hardening step, which even now is commonly used by pre-elite seed potato
producers. Plants bacterized in vitrowith Pseudomonas fluorescensstrains CHA0 and
IP10 were found to have a significantly higher fresh shoot weight compared to non-
bacterized plants in the same system. Arbuscular mycorrhizal (AM) fungi have also
been shown to reduce drought stress and increase disease resistance.

7.6 Co-inoculation strategies

In recent years, a number of studies on co-inoculation of two or more benefi-

cial microorganisms for better crop productivity have been reported. Positive effects,
such as increase in biomass parameters, nitrogen-content and yield have been found in
legumes inoculated with Rhizobiumand Azospirillum. These positive effects may be
attributed to early and increased nodulation, enhanced N2fixation rates, and a general
improvement of root development. Stimulation of nodulation following the inoculation
with Azospirillummay be derived from an increase in production of lateral roots, root
hair density and branching, but also from the differentiation of a greater number of
epidermal cells into root hairs susceptible for infection by rhizobia. Nodulation by rhi-
zobia co-inoculated with Azospirillummay also be enhanced by an increased secretion
of root flavonoid substances that are involved in the activation of the nodulation genes
in Rhizobium(Dobbelaere et al., 2001). Considerable results have also been obtained
on grain yield, N, P, K content in wheat coinoculated with Azospirillum brasilenseand
Rhizobium meliloti (Askary et al., 2009).

36
Soil Microbes and their interaction with plants

7.7 Bioremediation and phytoremediation

Phytoextraction, actuated by hyperaccumulating or non-hyperaccumulating
species, could be improved by using a plant-microbe system (Zhuang et al. 2007), thus
contributing to novel promising methods for the cleaning-up of soils contaminated by
heavy metals. Rhizobacteria of the genus Azospirillumhave been extensively used for
crop phytostimulation as above stated, thanks to the positive interaction betweenbacte-
ria and plants at root level (Dobbeleare et al., 2001; Dobbeleare et al., 2003; Russo et
al., 2005; Russo et al., 2008). The implementation of lead phytoextraction in contami-
nated industrial soils by applying A. brasilense Sp245 to plants of indigenous species
belonging to Mediterranean forestry was investigated. The possible phytoextraction
ability was evaluated in Myrtus communis L. and Laurus nobilisL., previously se-
lected among other plant species that were found able to grow in the contaminated
areas, on the basis of the Pb content (Emission Spectrophotometer Atomic Plasma,
ICP-AES), the growing speed and the vegetative habitus. By trials carried out in green-
house, it was shown that A. brasilense Sp245 can enhance the plant growth in Pb con-
taminated soil and affect the plant total lead content. Greenhouse trials were performed
for 2 and 9 months, and plants were grown in pot in the presence of two level of Pb (312
and 4345 ppm). The presence of Sp245 positively affected the total amount of Pb that
was removed by plants, either as total biomass produced (Figure 1) or as specific Pb
concentration, as a consequence of the incremented root growth, attesting the synergic
effect of plants and microorganisms in a bioremediation system, and ashigher specific
Pb concentration. Moreover the bioconcentration factor (Pb in plant tissues/Pb in soil)
and translocation factor (Pb in leaves and shoots/Pb in roots) were significantly af-
fected by the presence of A. brasilenseSp245, attesting the synergetic effect of plants
and microorganisms in a rhizoremediation system.

Figure 12. Effect of Azospirillum brasilenseSp245 inoculation on root system in Lau-

rus nobilisand Myrtus communisafter nine months of pot cultivation with Pb polluted
soil.
37
 plant- pathogen interaction

Conclusion:

Competition between plants is high, because plant growth in natural environ-

ments frequently takes place in dense stands of established vegetation. Thus both, ben-
eficial and deleterious associations of plants with micro-organisms and the regulation
of these associations by soil faunal activity may be of fundamental importance for
individual plant species, but also for the species composition of the whole community.

Mycorrhizas, the symbioses established between plant roots and soil fungi, rep-
resent a vital component in plant ecosystems. The composition and pattern of root
exudates affect microbial activity and population numbers. Plant species, plant devel-
opmental stage and soil type have been indicated as major factors determining the com-
position of rhizosphere microbial communities. As shown in many studies, there is no
general decision about the key player: the diversity and predominance of rhizosphere
microbial population depend on a number of abiotic and biotic factors of a particular
ecological niche.

The understanding of interactions between bacteria and fungi living in the my-
corrhizosphere is essential for describing the soil-plant interface. In recent advances in
molecular biology and genome sequencing have provided tools to address questions
about the identity and role of bacteria associated with mycorrhizal fungi. And Endo-
bacteria are consistently present in AM fungi, providing a sound demonstration of the
tripartite nature of mycorrhizas.

In sum, this can constitute overwhelming evidence indicating that an ever ex-
ploitation of plant growth promoting rhizobateria (PGPR) can be a true success story in
sustainable agriculture. As a consequence, current production methods in agriculture,
e.g., the improper use of chemical pesticides and fertilizers creating a long list of envi-
ronmental and health problems, will reduce.

38
Soil Microbes and their interaction with plants

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