Communication in the Rhizosphere, a Target

for Pest Management

Juan Antonio López-Ráez, Harro Bouwmeester, and Maria J. Pozo

Abstract The industrial agriculture has given rise to an excessive use and misuse
of agrochemicals causing environmental pollution. Therefore, it is urgent to find
alternatives that are more environmentally friendly than chemical fertilizers and
pesticides for disease control. The key to achieve successful biological control strat-
egies is the knowledge of the ecological interactions that occur belowground. The
rhizosphere constitutes a very dynamic environment harbouring the plant roots and
many organisms. Plants communicate and interact with those organisms through the
production and release of a large variety of secondary metabolites into the rhizo-
sphere. Thus, they use these metabolites to defend themselves against soil-borne
pathogens, which can adversely affect plant growth and fitness, but also to establish
mutualistic associations with beneficial soil microorganisms. However, despite the
importance of these plant-organism interactions the mechanisms regulating them
remain largely unknown.
We review here chemical communication that takes place in the rhizosphere
between plants and other soil organisms, and the potential use of this molecular
dialogue for developing new biological control strategies against deleterious organ-
isms. We focus on the knowledge of the root parasitic weed germination stimulants –
strigolactones – to develop more efficient control methods against this pest.

J.A. López-Ráez (* s-*0OZO

Department of Soil Microbiology and Symbiotic Systems,
Estación Experimental del Zaidín (CSIC), Profesor Albareda 1, 18008 Granada, Spain
e-mail: juan.lopezraez@eez.csic.es; mariajose.pozo@eez.csic.es
H. Bouwmeester
Laboratory of Plant Physiology, Wageningen University,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
Center for Biosystems Genomics, Wageningen, The Netherlands
e-mail: harro.bouwmeester@wur.nl

E. Lichtfouse (ed.), Agroecology and Strategies for Climate Change, 109

Sustainable Agriculture Reviews 8, DOI 10.1007/978-94-007-1905-7_5,
© Springer Science+Business Media B.V. 2012
110 J.A. López-Ráez et al.

Finally, we illustrate this with an exciting example: the use of the mutualistic
arbuscular mycorrhizal symbiosis for controlling root parasitic weeds by reducing
the production of strigolactones in the host plant.

Keywords 2HIZOSPHERE s #HEMICAL COMMUNICATION s 3IGNALLING s "IOLOGICAL

CONTROL s 3TRIGOLACTONES s !RBUSCULAR MYCORRHIZAL FUNGI s 2OOT PARASITIC PLANTS s
Soil-borne pathogens

Abbreviations
AM arbuscular mycorrhiza
AHL N-acyl homoserine lactone
PGPF plant growth promoting fungi
PGPR plant growth promoting rhizobacteria
QS Quorum sensing

1 Introduction

Plants are living organisms that continuous and reciprocally communicate with
other organisms in their environment. However, unlike animals plants cannot
speak, see, listen or run away, and therefore they largely rely on chemicals as sig-
nalling molecules to perceive environmental changes and survive. Thus, plants use
flower colour and volatiles to attract pollinators, use chemicals to defend them-
selves against enemies such as pathogens and herbivores, but they also use signal-
ling molecules to establish mutualistic beneficial associations with certain
microorganisms such as bacteria and fungi (Fig. 1). Microorganisms can affect
plant growth and development, change nutrients dynamics, susceptibility to dis-
ease, tolerance to heavy metals, and can help plants in the degradation of xenobiot-
ics (Morgan et al. 2005). As a result, these plant-microorganism interactions have
considerable potential for biotechnological exploitation. A nice example of this
complex and precisely regulated signalling takes place underground, where plants
use the roots to communicate and interact with other organisms in the so-called
rhizosphere.
The term rhizosphere derives from the Greek words rhiza, which means root, and
sphere, meaning field of influence (Morgan et al. 2005). The rhizosphere is the nar-
row soil zone surrounding plant roots that contains a wide range of organisms and
is highly influenced by the roots, the root exudates and by local edaphic factors
(Bais et al. 2006; Badri et al. 2009). Originally, the root system was thought only to
provide anchorage and uptake of nutrients and water. However, it has been shown
that roots are chemical factories that mediates numerous underground interactions
Communication in the Rhizosphere, a Target for Pest Management 111

Fig. 1 Plant interactions with other organisms. Positive and negative interactions occurring
aboveground and belowground in the rhizosphere. Yellow arrows indicate root exudates (Adapted
from Pozo and Azcón-Aguilar 2007)

(Badri et al. 2009). Plants produce and exude through the roots a large variety of
chemicals including sugars, amino acids, fatty acids, enzymes, plant growth regulators
and secondary metabolites into the rhizosphere some of which are used to commu-
nicate with their environment (Siegler 1998; Bertin et al. 2003; Bais et al. 2006).
Moreover, the release of root exudates together with decaying plant material provides
carbon sources for the heterotrophic soil biota. On the other hand, microbial activity
in the rhizosphere affects rooting patterns and the supply of available nutrients to
plants, thereby modifying the quantity and quality of root exudates (Barea et al.
2005). Of special interest in this rhizosphere communication are the so-called
secondary metabolites, which received this name because of their presumed secondary
importance in plant growth and survival (Siegler 1998). These metabolites include
compounds from different biosynthetic origins and have been shown to be of eco-
logical significance because they are important signals in several mutualistic and
pathogenic plant-organism interactions (Estabrook and Yoder 1998; Siegler 1998;
Bertin et al. 2003; Bais et al. 2006).
112 J.A. López-Ráez et al.

2 Interactions in the Rhizosphere

In the rhizosphere some of the most complex chemical, physical and biological
interactions between plant roots and other organisms occur influencing plant fitness.
Among these relationships we can find root-root, root-microbe and root-insect
interactions. Many of these interactions have a neutral effect on the plant. However,
the rhizosphere is also a playground for beneficial microorganisms establishing
mutualistic associations with plants, and a battlefield for soil-borne pathogens which
establish parasitic interactions (Raaijmakers et al. 2009).

2.1 Parasitic Interactions

As mentioned above, the rhizosphere is not only the playground for mutualistic
associations, but also a battlefield where parasitic interactions between plants and
soil-borne pathogens take place (Raaijmakers et al. 2009). In most agricultural eco-
systems, these negative interactions are economically important as they cause
important limitations in the production of marketable yield. It has long been under-
stood that the development of disease symptoms is not solely determined by the
pathogen responsible, but is also dependent on the complex interrelationship
between host, pathogen and prevailing environmental conditions. Negative interactions
with plant roots include pathogenesis by bacteria, true fungi or oomycetes, inverte-
brate herbivory and parasitism between plants (Agrios 2005; Bais et al. 2006).
Among them, fungi and oomycetes, nematodes and parasitic plants are major players
in the rhizosphere exerting a serious threat to world agricultural production.
Comparatively, fewer bacteria are considered as soil-borne plant pathogens, with
some exceptions such as Ralstonia solanacearum (causing bacterial wilt of tomato),
the enteric phytopathogen Erwinia carotovora, responsible of the bacterial soft rot,
and Agrobacterium tumefaciens, the causal agent of crown gall disease (Hirsch
et al. 2003; Genin and Boucher 2004; Badri et al. 2009).

2.1.1 Fungi and Oomycetes

Soil-borne fungal plant pathogens are important determinants in the dynamics of

plant populations in natural environments and in agriculture. Fungi and oomycetes
are the most important soil-borne microbial plant pathogens, causing economically
important losses. They can cause complete destruction of plants and even the total
loss of yield (Otten and Gilligan 2006). More than 8,000 species of fungi are known
to cause diseases of plants, and most plants are susceptible to several fungal patho-
gens. The majority of soil-borne fungi are necrotrophic, implying that they do not
require a living cell to obtain nutrients. They normally use enzymes and toxins to
kill host tissue before hyphal penetration and infection. The most harmful root
Communication in the Rhizosphere, a Target for Pest Management 113

pathogenic fungi include the genera Fusarium spp, Verticillium spp and Rhizoctonia
solani, which affect crops such as barley, wheat, maize, potato and tomato all over
the world (Priest and Campbell 2003; Garcia et al. 2006).
The oomycetes include a unique group of biotrophic and hemibiotrophic plant
pathogens that gain their nutrients from living cells, and are considered as non-
true fungi. Indeed, although they are physiologically and morphologically simi-
lar to fungi they belong to different phylogenetical groups. The oomycetes are
phylogenetically more closely related to brown algae than to fungi and, in contrast
to fungi, they contain cellulose in their cell wall instead of chitin (Raaijmakers
et al. 2009). However, despite being only distantly related to fungi, the oomy-
cetes have developed very similar infection strategies. These pathogens estab-
lish intimate relations with their hosts by forming an organ called haustorium,
which is used to obtain nutrients from the plant, redirecting host metabolism
and suppressing host defences. The oomycetes include some of the most destruc-
tive plant pathogens worldwide, particularly in the genera Phytophthora and
Phytium, that affect important crops such as potato, tomato, lettuce and soybean
(Raaijmakers et al. 2009).

2.1.2 Nematodes

Nematodes are small and complex worm-like eukaryotic invertebrates that rank
among the most numerous animals on the planet (Perry and Moens 2006). Most
nematodes in soil are free-living and consume bacteria, fungi and other nematodes,
but some can also parasitize plant roots being important crop pests in agricultural
ecosystems. Some feed on the outside of the root (ectoparasites), some penetrate
and move inside the root (endoparasites), and some set up a feeding site in the inte-
rior of the root and remain there for reproduction (sedentary endoparasites). Upon
infection, nematodes cause important changes in root cells in order to complete
their life cycle. Although the parasitism is rarely fatal for the infected plant, there
are substantial consequences of the interaction such as stunted growth, chlorosis and
poor yields. The most economically important groups of nematodes are the seden-
tary endoparasites, which include the genera Meloidogyne (root-knot nematodes)
and Heterodera and Globodera (cyst nematodes). They are particularly important
in tropical and subtropical regions (Bird and Kaloshian 2003; Williamson and
Gleason 2003).
Root-knot nematodes are obligate biotrophic pathogens found in all temperate
and tropical areas that have evolved strategies for infesting thousands of plant species
such as cereals, tomato, potato and tobacco (Caillaud et al. 2008). These root patho-
gens must locate and penetrate a root, migrate into the vascular cylinder and estab-
lish a permanent feeding site, known as giant cells. Unlike root-knot nematodes,
cyst nematodes are only able to infect a few plant species, principally soybean and
potato, and are more destructive as they migrate and travel intracellularly through
the root (Fuller et al. 2008). In both cases, these events are accompanied by exten-
sive signalling between the nematode and the host.
114 J.A. López-Ráez et al.

Parasitic plant

Strigolactone
O O O O O O O O O O
O O O O O O O O O O O O O O
O O O O O O O O O O O O O O O O O O O O O
O O O O O O O O O O O O O O O O O O O O O O O O O O O O
O O O
O O O O O O O O O
O O O O O O O O O O O O O O O O O O O O O O O O O
O O
O O
O O O O O O O O O O O O O O O O O
O O O O O O O O O
O O O
O O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O
O O O O O O O O O O O O O O O
O O O O O O O O O O O O

Parasitic plant seed Haustorium

    

Fig. 2 Life cycle of root parasitic plants. (a) Seeds are buried in the soil and perceive the germination
stimulants exuded by the roots of the host plant, strigolactones, and germinate. (b) The germinated
seeds form a haustorium by which they attach to the host root, establishing a xylem-xylem connection.
(c) The parasitic plant develops, and the shoots emerge from the soil. There is areduction of host
growth. (d) Parasitic plant flowering and crop yield reduction. (e) Production of mature seeds that
end up in a new generation of seeds in the soil (Redrawn from Sun et al. 2007)

2.1.3 Root Parasitic Plants

Root parasitic plants of the family Orobancheaceae, including the Striga, Orobanche
and Phelipanche genera are some of the most damaging agricultural pests, causing
large crop losses. These obligate root parasites attach to the roots of many plant spe-
cies and acquire nutrients and water from their host through a specialized organ
called haustorium (Estabrook and Yoder 1998; Bouwmeester et al. 2003). Striga is
a hemiparasite, which means that it obtains nutrients from its host but it can also
perform its own photosynthesis. It infects important crops such as maize, sorghum,
pearl millet, finger millet and upland rice, causing devastating losses in cereal yields
in Africa (Gressel et al. 2004). On the other hand, the holoparasitic (lacking chloro-
phyll and being completely dependent on their host) Orobanche and Phelipanche
spp. affect important agricultural crops in more temperate climates such as southern
Europe, Central Asia and the Mediterranean area parasitizing legumes, tobacco,
crucifers, sunflower and tomato (Joel et al. 2007).
Although root parasitic plants parasitize different hosts in different parts of the
world, their lifecycles are very similar and involve germination in response to a root
host stimulus, radicle growth towards the host root, and attachment and penetration
through the haustorium (Fig. 2). Upon vascular connection, the parasitic plant
obtains nutrients and water from the host plant, negatively affecting plant fitness and
crop yield. After emergence from the soil, parasitic plants will flower and produce
new ripe seeds that are shattered increasing the seed bank (Fig. 2) (Bouwmeester
et al. 2003; López-Ráez et al. 2009). Parasitic weeds are difficult to control
because most of their life cycle occurs underground and therefore new control strat-
egies that focus on the initial steps in the host-parasite interaction are required
(López-Ráez et al. 2009).
Communication in the Rhizosphere, a Target for Pest Management 115

2.2 Mutualistic Beneficial Associations

The rhizosphere generally helps the plant by maintaining the recycling of nutrients,
providing resistance to diseases and to improve tolerance to toxic compounds. When
plants lack essential mineral elements, such as phosphorous or nitrogen, symbiotic
relationships can be beneficial and promote plant growth. Thus, plants biologically
interact with other organisms to establish mutualistic associations which rely on a
mutual fitness benefit. Mutualism is very ancient, indeed is thought to have driven
the evolution of much of the biological diversity present today (Thompson 2005).
In addition, mutualism plays a key role in ecology being very important for the
correct functioning of the terrestrial ecosystem. Microorganisms that positively
affect plant growth and health include the plant growth-promoting rhizobacteria
(PGPR) and plant growth-promoting fungi (PGPF), the nitrogen-fixing Rhizobium
bacteria (rhizobia), and the mycorrhizal fungi (mycorrhiza). The PGPR are non-
symbiotic beneficial rhizosphere bacteria that are known to participate in many
important ecosystem processes, such as nitrogen fixation, nutrient cycling, seedling
growth, phytohormone production, and biological control of plant pathogens (Barea
et al. 2005; Raaijmakers et al. 2009). The most commonly genera described as including
PGPR are Pseudomonas and Bacillus. The PGPF include rhizospheric non-symbiotic
beneficial fungi from the Deuteromycetes, e.g. Trichoderma, Gliocladium and non-
pathogenic Fusarium oxysporum (Raaijmakers et al. 2009). These ubiquitous soil
fungi are effective in controlling a broad range of phytopathogenic fungi by competi-
tion, antibiosis and mycoparasitism (Raaijmakers et al. 2009).
Other beneficial microorganisms, the endophytes, establish mutualistic symbio-
sis with plants by colonizing the root tissues and promote plant growth and plant
protection (Barea et al. 2005). Although new endophytic microbes which colonize
roots and promote plant growth are being found such as the fungus Piriformospora
indica (Varma et al. 1999), the best studied examples of rhizosphere mutualism are
those established with rhizobia bacteria and mycorrhizal fungi.

2.2.1 Rhizobia

Rhizobia are free-living soil bacteria which colonize plant roots (endosymbionts)
establishing a mutualistic relationship with most of the plant legume species world-
wide (Sprent 2009). The two partners cooperate in a nitrogen-fixing symbiosis of
major ecological importance because in many environments nitrogen limits plant
growth (Masson-Boivin et al. 2009). Legume-rhizobia symbiosis is a classic exam-
ple of mutualism, where rhizobia supply ammonia (NH4+) or amino acids to the
plant and in return receive organic acids (principally malate and succinate) as a
carbon and energy source, proteins and sufficient oxygen to facilitate the fixation
process (Fig. 3a). Fixed nitrogen is a limiting nutrient in most environments, with
the main reserve of nitrogen in the biosphere being the molecular nitrogen in the
atmosphere. Molecular nitrogen cannot be directly assimilated by plants, but it
116 J.A. López-Ráez et al.

Rhizobia Flavonoid
Nodule
. . .
. . . . . . . . .
. .
. . . . . . . .
Nod factor
  

Strigolactone Mycelium

O O . O O
O O O O
OO O O O OO O O O O O O O
O O OO O O O OO O
. . . .
. OO O . OO O OO O . OO O
OO O OO O .
O O O O O O O O O O O O O O O O
. . . .
OO O OO O OO O OO O OO O OO O OO O OO O
O O . O O . . .
. . . . . . . .
OO O OO O

AM fungus spore AM hyphae Arbuscule

1 2 3 4

Fig. 3 Scheme of signalling and establishment of plant-microorganism mutualistic associations in

the rhizosphere. (a) Molecular signalling and nodulation process during rhizobia-legume association;
1 Production of flavonoids by the host plant under low nitrogen conditions. 2 Flavonoids are per-
ceived by the bacteria and induce the production of the bacterial Nod factors. 3 Nod factors are rec-
ognized by the host plant and initiate the symbiotic program for nodulation. (b) Molecular signalling
and mycorrhizal establishment between plants and arbuscular mycorrhizal (AM) fungi; 1 Under
phosphate deficient conditions plants release the signalling molecules strigolactones. 2 Strigolactones
are perceived by germinating spores of AM fungi and induce hyphal branching and growth towards
the host root. 3 AM hyphae produce Myc factors which are perceived by the host plant initiating the
symbiotic program. 4 AM fungus colonizes the host root forming arbuscules and develops an exter-
nal mycelia network. AM symbiosis and nodulation increase plant fitness and crop production

becomes available through the biological nitrogen fixation process that only some
prokaryotic cells (diazotrophs), including rhizobia, have developed (Masson-Boivin
et al. 2009).
In rhizobial plants, nitrogen fixation takes place in special organs known as
nodules. On the roots of host plants, principally in the root hairs, rhizobia colonize
Communication in the Rhizosphere, a Target for Pest Management 117

intracellularly by triggering the formation of an infection thread structure that

elongates, ramifies and penetrates inside the emerging nodule. Then, they become
internalized in plant cells via an endocytosis-like process. Once reached the central
tissue, known as symbiosome, release the rhizobia in these cells where they multi-
ply and differentiate morphologically into bacteroids. Within the nodules the host
plant provides the bacteria with the carbohydrates they need. In return, the bacte-
roids by the action of the enzyme nitrogenase fix the atmospheric nitrogen from the
atmosphere into a plant usable form (NH4+). Then, the NH4+ is converted into amides
or ureides which are translocated to the plant xylem (Masson-Boivin et al. 2009).

2.2.2 Mycorrhiza

Fungi are eukaryotic, filamentous, multicellular and heterotrophic organisms that

produce a network of hyphae called mycelium which absorbs nutrients and water
from the surrounding substrate. Mycorrhiza is a symbiotic, generally mutualistic
symbiosis established between certain soil fungi and the roots of most vascular
plants, including agricultural and horticultural crop species (Smith et al. 2006;
Smith and Read 2008). Mycorrhizas are commonly grouped into two categories
based on their colonization style. They are considered either ectomycorrhiza if they
colonize host plant roots extracellularly or endomycorrhiza, if they colonize intrac-
ellularly. Ectomycorrhiza are typically formed between the roots of around 10% of
plant families, mostly woody plants, and fungi belonging to the Basidiomycota,
Ascomycota, and Zygomycota (Bonfante and Genre 2010). Ectomycorrhizas con-
sist of a hyphal sheath, or mantle covering the root tip and a hartig net of hyphae
surrounding the plant cells within the root cortex. Endomycorrhiza include the
arbuscular mycorrhizal (AM) symbiosis, the ericoid mycorrhiza established with
members of the family Ericaceae and the orchid mycorrhiza, a symbiotic relation-
ship between fungi and the roots of plants of the family Orchidaceae (Smith and
Read 2008).
Among them, the AM symbiosis is the most common form of mycorrhizal sym-
biosis and consists of an association established between certain soil fungi of the
phylum Glomeromycota – which is widely distributed throughout the world – and
over 80% of terrestrial plants, including most agricultural and horticultural crop
species (Smith et al. 2006; Parniske 2008). This association is considered to be
older than 400 million years and it has been postulated to be a key step in the evolu-
tion of terrestrial plants (Smith et al. 2006). AM fungi are obligate biotrophs and
therefore they depend entirely on the plant to complete their life cycle (Fig. 3b).
They colonize the root cortex forming specialized and tree-like subcellular struc-
tures called arbuscules for nutrient exchange (Parniske 2008). Through the symbiosis,
the fungus obtains carbohydrates from the host plant for which, in return, the fungus
assists the plant in the acquisition of mineral nutrients (mainly phosphorous) and
water, hence improving plant fitness (Fig. 3b). AM symbiosis gives rise to the for-
mation of mycorrhizal networks that offer a number of advantages for the acquisi-
tion of nutrients such as fungal hyphae extension beyond the area of nutrient
depletion and increase of the surface area for the absorption of nutrients. Moreover,
118 J.A. López-Ráez et al.

some mycorrhizal fungi can access forms of nitrogen and phosphorous that are not
available to non-mycorrhizal plants, for example when bound in organic forms
(Morgan et al. 2005). Thus, AM symbiosis contributes to global phosphate and
carbon cycling and influences primary productivity in terrestrial ecosystems (Fitter
2005). Besides improving the nutritional status, the symbiosis enables the plant to
perform better under stressful conditions (Pozo and Azcón-Aguilar 2007; Parniske
2008). Therefore, AM symbiosis plays a crucial role in agriculture and natural
ecosystems.
In summary, the rhizosphere is an environment influenced by the plant root
exudates where both pathogenic and beneficial interactions between plant and other
organisms constitute a major influential force on plant growth and fitness, soil quality
and ecosystem dynamics.

3 Molecular Dialogue in the Rhizosphere

All the different interactions reported above are based on molecular communica-
tion occurring belowground. Plants produce and release enormous amounts of
chemicals into the rhizosphere through their roots in order to communicate and
interact with their environment. Root exudates can be divided into two classes of
compounds: high-molecular weight such as polysaccharides and proteins, and low-
molecular weight compounds including amino acids, organic acids, sugars, pheno-
lics, and other secondary metabolites (Bais et al. 2006). Although the functions of
most of the compounds present in root exudates have not been determined so far, it
has been determined that several of them are essential to establish plant interac-
tions with other organisms in the rhizosphere. Equally, chemical signals secreted
by the rhizospheric organisms are also involved in early steps of host recognition
and colonization, and necessary for the establishment of the association. These
signalling molecules are important in both negative and positive interactions (Bais
et al. 2006).

3.1 Communication Between Plants and Parasites

In plant-plant interactions, plants secrete phytotoxins such as the flavonoids cathe-

chins and benzoflavones, and sorgoleone that reduce the establishment, growth, or
survival of susceptible plant neighbours, a phenomenon known as allelopathy, from
the Greek words allele (mutual) and pathy (harm or suffering), to avoid competition
with other plant species (Weir et al. 2004). Plants also produce germination stimulants
of seeds the root parasitic plants of the genera Orobancheaceae – strigolactones –
which are essential for the establishment of a negative association (Bouwmeester
et al. 2003) (see Sect. 6).
Communication in the Rhizosphere, a Target for Pest Management 119

In addition to plant-plant interactions, plants produce and exudate antimicrobial

secondary metabolites such as indole, terpenoids, phenylpropanoids including the
flavonoids (e.g. rosmarinic acid) which show potent antimicrobial activity against
an array of soil-borne pathogens (Bais et al. 2002). Antimicrobial compounds can
be classified in two different classes: phytoanticipins, which occur constitutively in
healthy plants acting as chemical barriers to fungal pathogens, and phytoalexins,
including terpenoids, glycosteroids and alkaloids, that are induced in response to
pathogen attack but not normally present in healthy plants (Badri et al. 2009). It has
been described that phenylpropanoid levels, a diverse family of organic compounds
derived from the amino acid phenylalanine which provide protection against herbi-
vores and pathogens, were significantly higher in roots that were challenged by non-
host bacterial pathogens Pseudomonas syringae strains compared to host bacterial
strains (Bais et al. 2006). Bacterial pathogens able to infect roots and cause disease
were resistant to these compounds, suggesting an important role of phenylpropanoids
in defense against non-host pathogens (Bais et al. 2006).
Besides functioning as antimicrobial compounds, some secondary metabolites
can act as chemoattractants for certain pathogens. For example, it has been shown
that before infection establishment, zoospores of the pathogen oomycete
Phytophthora sojae are chemically attracted by the isoflavones daidzein and
genistein secreted by soybean roots (Hirsch et al. 2003). In a large number of patho-
genic bacteria, initiation of the production and secretion of virulence factors is con-
trolled by a phenomenon known as quorum-sensing (QS). QS is a cell-cell
communication and density-dependent regulatory mechanism which is mainly
induced by the small molecules N-acyl homoserine lactones (AHLs) (Bais et al.
2006). The rhizosphere contains a higher proportion of AHL-producing bacteria
than bulk soil, suggesting that they play a role in colonization (Bais et al. 2006). It
has been also suggested that plants could be using root-exuded compounds in the
rhizosphere to take advantage of this bacterial communication system and influence
colonizing communities.
On the other hand, the association between plants and nematodes is also sub-
jected to an extensive signalling between the nematode and its host, although the
knowledge of the initial signalling molecules is scarce. It was shown that hatching
of juveniles of the cyst nematode Globobera is controlled by solanoeclepin A, a
molecule secreted by the roots of some Solanaceae species such as potato and
tomato (Schenk et al. 1999). More recently, it has been reported that Medicago roots
released a volatile (dimethyl sulphide) that attract nematodes (Horiuchi et al. 2005).
Reciprocally, nematodes secrete cytokinins that play a role in cell cycle activation
and in establishing the feeding in the host root.

3.2 Chemical Signalling Between Plants and Mutualists

A functional mutualistic relationship also implies and requires a signal exchange

between both partners that leads to mutual recognition and development of symbiotic
120 J.A. López-Ráez et al.

structures (Siegler 1998). Moreover, this molecular dialogue must be precisely

regulated in order to avoid opportunities for malevolent organisms (Hirsch et al.
2003; Bouwmeester et al. 2007). An important number of plant-derived signalling
molecules destined to the establishment of beneficial associations with soil microbe
partners belong to the class of the secondary metabolites (Siegler 1998; Steinkellner
et al. 2007). We will go into detail on a number of them, the flavonoids and the
strigolactones, which are key signalling molecules in the interaction rhizobia-
legume and in AM symbiosis, respectively.

3.2.1 Communication in Nodulation

Rhizobia-legume symbiosis is an ecologically important mutualistic association

because of its implication in nitrogen fixation. The establishment of this symbio-
sis requires a high degree of coordination between the two partners, which is
based on a finely regulated molecular dialogue that orchestrates the complex sym-
biotic program (Garg and Geetanjali 2007; Badri et al. 2009) (Fig 3a). The chemi-
cal communication between the plant and the bacteria starts, before there is any
contact between the partners, with the production of flavonoids and isoflavonoids
by the host plant (Badri et al. 2009; Faure et al. 2009). More than 4,000 different
flavonoids have been identified in vascular plants, but just a small subset of them
are involved in this mutualistic interaction (Bais et al. 2006). Host legume-derived
flavonoids and related compounds act as chemo attractants for rhizobial bacteria
and as specific inducers of rhizobial nodulation genes (nod genes), that encode the
biosynthetic machinery for a bacterial signal – the lipochitooligosaccharides –
that are released into the rhizosphere (Fig. 3a) (Perret et al. 2000; Reddy et al.
2007). These bacterial-derived signals are known as Nod factors, and are specific
for different rhizobial strains. In turn, Nod factors are perceived by the host plant
which leads to the induction of root hair deformation and several cellular responses
such as ion fluxes. This mutual recognition precedes the intracellular infection by
the bacteria through the deformed root hair. The infection triggers cell division in
the cortex of the root where a new organ, termed nodule, appears as a result of
successive processes (Fig. 3a). Finally, in the nodule nitrogen fixation takes place
by the bacteria (see Sect. 2.2.1). Interestingly, this signal-detection machinery for
interaction with beneficial rhizobia employs common components that are also
implicated in the detrimental association with root-knot nematodes (Weerasinghe
et al. 2005).

3.2.2 Signalling During Arbuscular Mycorrhizal Symbiosis

In addition to their involvement in the rhizobia-legume symbiosis, flavonoids play a

role also in the AM symbiosis, which is also of ecological importance because it
contributes to phosphate and carbon cycling. Flavonoids act as stimulants of AM
Communication in the Rhizosphere, a Target for Pest Management 121

fungi hyphal growth, differentiation and root colonization (Steinkellner et al. 2007).
Besides flavonoids, the strigolactones, a recently described new class of plant
hormones regulating plant architecture (Gomez-Roldan et al. 2008; Umehara et al.
2008; Koltai et al. 2010; Ruyter-Spira et al. 2011), have been shown to be crucial for
a successful root colonization by the AM fungi (Akiyama et al. 2005). Interestingly,
strigolactones are present in the root exudates of a wide range of plants and trigger
a response only in AM fungi, not in other beneficial fungal species such as
Trichoderma and Piriformospora (Steinkellner et al. 2007).
AM fungi depend entirely on their plant host to complete their life cycle (Fig. 3b).
As for the rhizobia-legume association, AM symbiosis establishment and function-
ing also require a high degree of coordination between the two partners (Paszkowski
2006; Hause et al. 2007; Requena et al. 2007). The AM fungi-plant host molecular
dialogue starts in the rhizosphere with the production of strigolactones by the host
plant that induce hyphal branching in germinating spores of AM fungi (Akiyama
et al. 2005; Besserer et al. 2006; Parniske 2008). Spores of AM fungi can germinate
spontaneously and undergo an initial asymbiotic stage of hyphal germ tube growth,
which is limited by the amount of carbon storage in the spore. If a partner is nearby,
the hyphal germ tube grows and ramifies intensively through the soil towards the
host root (Bouwmeester et al. 2007). It has been suggested that these signalling
molecules, later known as strigolactones, may also act as the chemoattractant that
directs the growth of the AM hyphae to the roots (Sbrana and Giovannetti 2005).
Once the host-derived strigolactones are perceived by the fungus, it engages its
catabolic metabolism which results in hyphal branching that will increase the prob-
ability to contact the root and establish symbiosis. Similarly to the nodulation
process, it has been proposed that a Myc factor analogous to the rhizobial Nod factor
and produced by the metabolic active fungus, induces molecular responses in the
host root required for a successful AM fungal colonization (Fig. 3b) (Kosuta et al.
2003; Bucher et al. 2009). The chemical nature of the elusive Myc factor, has
remained unknown for a long time. However, it has been recently shown to have
structural similarities with rhizobial Nod factors (Maillet et al. 2011).
Despite the importance of strigolactones in the initiation of AM symbiosis, it is
unknown whether they also play a role in subsequent steps of the symbiosis. Since
strigolactones are considered plant hormones and are ubiquitous in plants, it is
tempting to speculate about their involvement in other plant-microorganism interac-
tions in the rhizosphere. Indeed, it has been recently shown that strigolactones
positively affect nodulation, although their effect was not due to an effect on the
bacteria (Soto et al. 2010). Probably, this just represent the tip of the iceberg of
biological roles for the strigolactones, showing the biological and ecological importance
of these signalling compounds.
Thus, plants form associations – either beneficial or detrimental – with other
organisms in the rhizosphere. Interestingly, most of these interactions are facilitated
by a molecular dialogue between the host and the symbionts through chemical cues,
which are crucial for the establishment of these belowground associations. However,
although some of these signalling molecules have been identified there are still
many other unknown factors involved.
122 J.A. López-Ráez et al.

4 Regulation of Chemical Communication in the Arbuscular

Mycorrhizal Symbiosis

As mentioned above, one of the primary roles of AM fungi in the symbiotic

relationship with plants is the supply of water and mineral nutrients, mainly phos-
phorous and nitrogen (Harrison 2005; Karandashov and Bucher 2005; Yoneyama
et al. 2007). In many areas of the world the concentration or availability of these
essential mineral nutrients in the soil is low, which results in an important negative
impact on plant growth and fitness. Phosphorous, which is taken up from the soil as
phosphate, is one of the least available of all essential nutrients in soils because of
its low mobility, resulting in phosphate depletion in the rhizosphere. Moreover, the
majority of the applied phosphorus may be fixed in the soil due to the interaction
with other ions and hence be unavailable to plants (Raghothama 2000). Similarly,
nitrogen availability may be limited due to its loss through volatilization and leaching
(Delgado 2002).
In agreement with the important role of AM fungi in the acquisition of mineral
nutrients, it was observed that root exudates produced by plants grown under phos-
phate limited conditions are more stimulatory to AM fungi than exudates produced
under adequate phosphate nutrition (Nagahashi and Douds 2004). Moreover, it was
shown that phosphate and nitrogen deficiency have a significant stimulatory effect
on the production and exudation of strigolactones by plants (Yoneyama et al. 2007;
López-Ráez et al. 2008a). Yoneyama and co-workers have suggested that the
response of strigolactones production and exudation to nutrient availability varies
between groups of plant species (Yoneyama et al. 2007). Thus, legumes, that can
establish symbiosis with rhizobia and acquire nitrogen from root nodules, only
respond to phosphate deficiency with enhanced strigolactone production to attract
AM fungi, whereas in non-leguminous plant species such as tomato both phosphate
and nitrogen starvation enhance the production of strigolactones. The strigolactones
have been detected in the root exudates of a wide range of plant species including
mono- and dicotyledonous, indicating their broad spectrum of action and impor-
tance in nature (Bouwmeester et al. 2007; Yoneyama et al. 2008).
Future research is required to elucidate the mechanisms by which the chemical
signalling between plants and AM fungi is regulated to further optimize this benefi-
cial mutualistic association.

5 Strigolactones: Ecological Significance in the Rhizosphere

Strigolactones have been recognized as a new class of plant hormones that inhibits
shoot branching and hence controls above ground architecture (Gomez-Roldan
et al. 2008; Umehara et al. 2008). More recently, it has been suggested that they
also affect root growth and root hair elongation (Koltai et al. 2010; Ruyter-
Spira et al. 2011), which shows they are even more important components in the
Communication in the Rhizosphere, a Target for Pest Management 123

regulation of plant architecture than already postulated. Long before the discovery of
their function as plant hormones, the strigolactones were described as germination
stimulants for the seeds of root parasitic plants Striga, Orobanche and Phelipanche
spp (Cook et al. 1972; Bouwmeester et al. 2003) (see Sect. 2.1.3). They are pro-
duced and exuded into the rhizosphere by plants in very low amounts, can stimulate
the germination of these parasitic plants in nano- and pico-molar concentrations,
and are instable in a watery environment and in alkaline soils (Bouwmeester et al.
2007; Yoneyama et al. 2009; Zwanenburg et al. 2009). Strigolactones are derived
from the carotenoids (Matusova et al. 2005; López-Ráez et al. 2008a) and all the
strigolactones characterized so far are remarkably similar, showing a similar chemical
structure (Fig. 4) (Rani et al. 2008; Yoneyama et al. 2009; Zwanenburg et al. 2009).
The structural core of the molecules consists of a tricyclic lactone (the ABC-rings)
connected via an enol ether bridge to a butyrolactone group (the D-ring). It has been
suggested that the biological activity of the strigolactones resides in the enol ether
bridge, which can be rapidly cleavage in an aqueous and/or alkaline environment
(Yoneyama et al. 2009; Zwanenburg et al. 2009; Akiyama et al. 2010).
An intriguing question was why plants would produce compounds that have such
negative consequences (parasitiation by parasitic plants) for the plants themselves.
The answer to this question came only few years ago when Akiyama and co-workers
demonstrated that these secondary metabolites are involved in signalling between
plants and mutualistic AM fungi (Akiyama et al. 2005). We now know that under
nutrient deficient conditions plants increase the production of strigolactones to
attract AM fungi and establish a mutualistic relationship, but the parasitic weeds
have evolved a mechanism by which they can abuse this ‘cry for help’ plant signal
to establish a negative interaction (Bouwmeester et al. 2007) (Fig. 4). The ability to
develop AM symbiosis is of great advantage to plants and this, therefore, likely
explains why strigolactones are secreted by plants despite the possibility of being
abused by root parasitic plants.
Again, a better understanding about how strigolactone signalling is regulated and
the possible specificity of different strigolactones seems crucial to further evaluate
their importance in the plant-parasitic plant and plant-AM fungus interactions and
favor one against another.

6 Control Strategies Against Root Parasitic Plants

As mentioned in Sect. 2.1.3, root parasitic plants are a serious threat to agriculture
causing enormous crop losses worldwide. One of the reasons of their devastating
effect is that these parasitic weeds are difficult to control because most of their life-
cycle occurs underground (Fig. 2). This fact makes the diagnosis of infection diffi-
cult and normally only after irreversible damage has already been caused to the
crop. To date, a wide number of approaches such as hand weeding, crop rotation,
sanitation, fumigation, solarization and improvement of soil fertility are being used
to control root parasites without the desirable success (Joel et al. 2007; Rispail et al.
124 J.A. López-Ráez et al.

Myc factors strigolactones Par factors?

O O
C
A B
O O O
OH D

Hyphal branching Germination

factors stimulants

AM fungi Orobancheaceae
MUTUALISTIC INTERACTION PARASITIC INTERACTION

Fig. 4 Underground communication between plants, arbuscular mycorrhizal (AM) fungi and
parasitic plants. Plants produce and release strigolactones into the rhizosphere to communicate
with AM fungi in order to establish a mutualistic association. As a response, AM fungi release the
so-called Myc factors which are recognized by the host plant. However, strigolactones can be
abused by root parasitic plants of the family Orobancheaceae as an indicator of host presence,
resulting in seed germination and establishment of a parasitic interaction. Similarly to the AM
signal (Myc factor), it has been suggested that, in response to the strigolactones, the germinating
parasitic seeds would produce Par factors (Modified from Bouwmeester et al. 2007)

2007; Scholes and Press 2008), and the most efficient control method – fumigation –
is environmentally hazardous. Therefore, new methods for a more effective control
against these agricultural pests are required. Since the root parasites affect their host
from the moment they attach and exert the greatest damage prior to emergence (Joel
et al. 2007; Scholes and Press 2008), the development of more effective control
strategies should focus on the initial steps in the host-parasite interaction. Particularly
on the germination of the parasitic weed seed stage, which is trigged by the strigolac-
tones (Sun et al. 2007; López-Ráez et al. 2009). In this sense, two general approaches
to control root parasitic plants may be envisaged: through enhanced or reduced seed
germination.
Communication in the Rhizosphere, a Target for Pest Management 125

6.1 Control Through Enhanced Germination

6.1.1 Trap and Catch Crops

This strategy consists in the use of non-host plant species that produce germination
stimulants – strigolactones -, inducing suicidal germination of the parasite’ seeds.
Once germinated, the seeds cannot survive without a suitable host, hence causing a
reduction in the parasite seed bank (Zwanenburg et al. 2009). These trap and catch
crops can be resistant in a later stage of the parasite lifecycle – trap crops – or
harvested before the seeds of the parasite are shed – catch crops – (Bouwmeester
et al. 2003; Sun et al. 2007). The effectiveness of catch and trap crops could be
increased by the selection of cultivars overproducing germination stimulants (breeding)
or through molecular engineering of such overproduction. The latter can potentially
be achieved by overexpression of the rate-limiting enzymes from the strigolactone
biosynthetic pathway such as CCD7 or CCD8. In addition to the suicidal germination
induced by these catch and trap crops with enhanced production of strigolactones,
they could favour arbuscular mycorrhizal colonization in the host plant, with the
corresponding benefits on plant growth, fitness and yield.

6.1.2 Synthetic Germination Stimulants

An alternative strategy to controlling root parasitic plant infestation through the induc-
tion of suicidal germination is the use of synthetic germination stimulants. In this
sense, the application at very low concentrations of the strigolactone analogues GR24,
GR7 and Nijmegen-1 to Striga-infested soils resulted in reduction in the seed popula-
tion (Johnson et al. 1976; Wigchert et al. 1999). However, one of the limitations of this
approach is that these synthetic germination stimulants are rather unstable in the soil.
Therefore, more stable compounds or suitable formulations should be developed in
order to overcome these stability problems and increase their effectiveness.

6.2 Control Through Reduced Germination

Another approach to avoid root parasitic weed infection is based on the opposite
strategy, aimed at reducing seed germination. However, since strigolactones are also
AM hyphal branching factors and are involved in plant architecture, the conse-
quences for the AM fungal community in the soil and possible unwanted side-effects
on plant architecture should be carefully evaluated before following this approach.

6.2.1 Soil Fertilization

As mentioned above, plants grown under nutrient deficient conditions, specially

regarding phosphate and nitrogen, are more active in producing and exuding
126 J.A. López-Ráez et al.

strigolactones and, therefore, in inducing germination of root parasitic plant seeds

(Yoneyama et al. 2007; López-Ráez et al. 2008a). In many areas of the world,
the concentration or availability of these essential mineral nutrients are limited in
the soil, fact that has a significant impact on plant growth and health. Therefore, the
use of fertilisers not only would improve soil fertility, plant fitness and crop yield,
but also would reduce strigolactone production by the host plant and hence reduce
the infection by parasitic weeds (Fig. 2). Indeed, the application of phosphate to
phosphate-deficient soils significantly reduced the population and infestation of the
parasites Orobanche minor in red clover and P. aegyptiaca in tomato plants (Jain
and Foy 1992; Yoneyama et al. 2001). However, since strigolactone production in
response to nutrient availability differs between plant species, fertiliser rate and
composition should be carefully optimised depending on the crop, soil fertility and
possibly the parasitic weed species before using this strategy as a control method.

6.2.2 Chemical Inhibitors

Strigolactones are derived from the carotenoids (Matusova et al. 2005; López-Ráez
et al. 2008a). Therefore, herbicides that inhibit carotenoid biosynthesis such as
fluridone, norflurazon, clomazone and amitrole could be used in very low concen-
trations as a tool to reduce strigolactone production and ultimately parasitic seed
infection (López-Ráez et al. 2009; Jamil et al. 2010). Indeed, it was observed that
application of these inhibitors at concentrations that do not cause chlorophyll
bleaching to maize, sorghum, cowpea, rice and tomato strongly reduces strigolac-
tone production and in vitro germination of Striga hermonthica and Phelipanche
ramosa seeds by the exudates of the treated plants (Matusova et al. 2005; López-
Ráez et al. 2008a; Jamil et al. 2010). These results show that treatments with such
herbicides may be an effective and relatively cheap method to reduce parasitic
weed infestation in the field either alone or in combination with other control
strategies.

6.2.3 Breeding for Low Strigolactone Production Cultivars

The selection of cultivars with low production/exudation of strigolactones could be

an attractive strategy to control root parasitic weeds. In this sense, genetic variation
for the production of strigolactones has been observed for different crops. We have
shown that different cultivars of tomato produce different amounts of strigolactones
(López-Ráez et al. 2008b). The tomato mutant high pigment-2 (hp-2dg), which is an
important mutant line introgressed into commercial tomato cultivars because of its
enhanced levels of carotenoids including lycopene, was less susceptible to P. aegy-
tiaca infection than the corresponding wild-type, which correlated with a reduced
production of strigolactones (López-Ráez et al. 2008b). Genetic variation for low
germination stimulant production has been also described in sorghum, fact that was
used to breed for Striga resistant varieties and introduce them into high yielding
Communication in the Rhizosphere, a Target for Pest Management 127

sorghum cultivars in several African countries (Ejeta 2007). Therefore, selecting

programs to breed for cultivars with low strigolactone production is a valid and
promising strategy.

6.2.4 Genetic Engineering

Molecular biology techniques targeting one or more of the rate-limiting genes from
the strigolactone biosynthetic pathway could be another approach to reduce strigolac-
tone biosynthesis. Indeed, ccd7 and ccd8 mutants of several plant species show a
reduced production of strigolactones (Gomez-Roldan et al. 2008; Umehara et al.
2008). Moreover, genetic engineering using RNAi technology on CCD7 and CCD8
genes induced a significant reduction on strigolactones in tomato, which correlated
with a reduction in the germinating activity of P. ramosa seeds (Vogel et al. 2010;
Kohlen, López-Ráez and Bouwmeester, unpublished). Therefore, molecular engi-
neering may be an important and efficient component of a long-term strategy for
parasitic weed control. However, further research is required to completely charac-
terize the biosynthetic pathway of strigolactones in order to select appropriate target
genes with temporal or inducible promoters.

6.2.5 Use of Beneficial Microorganisms: Arbuscular Mycorrhizas

The fact that the strigolactones play a dual role in the rhizosphere as signalling mol-
ecules for both AM fungi and root parasitic plants (Fig. 4), and that AM symbiosis
greatly benefits plant fitness make the strigolactones a suitable candidate to develop
environmentally friendly biological control methods against parasitic weeds. Indeed,
it was shown that AM fungal inoculation of maize and sorghum led to a reduction
in Striga hermonthica infection in the field (Lendzemo et al. 2005), and it was pro-
posed that this reduced infection was caused, at least partially, by a reduction in the
production of strigolactones in the mycorrhizal plants (Lendzemo et al. 2007; Sun
et al. 2008) (Fig. 5). A similar effect was observed in pea, where AM colonization
reduced seed germination of Orobanche and Phelipanche species (Fernández-
Aparicio et al. 2010). We have recently shown that AM symbiosis in tomato also
leads to a reduction in the germination stimulatory activity of tomato exudates for
seeds of the parasite P. ramosa, and have analytically demonstrated that this reduction
is caused by a reduction in the production of strigolactones (López-Ráez et al.
2011). Moreover, we have also observed that this reduction requires a fully estab-
lished mycorrhizal association (López-Ráez et al. 2011). The results with maize,
sorghum, pea (although not analytically supported) and tomato suggest that the
reduction in strigolactone exudation induced by AM symbiosis is conserved across
the plant kingdom. As AM fungi colonize roots of most agricultural and horticul-
tural species and are widely distributed around the globe, this environmentally
friendly biocontrol strategy can potentially be used in the majority of economically
important crops that suffer from these root parasites worldwide. Thus, mycorrhizal
128 J.A. López-Ráez et al.

!-  !-

O O
O O
O O O O O O O
O O O O O O O
O O O O O O
O O O O O O O O
O O
O O O O O
O O O
O O O O O
O O
O O O O O
O O O
O O O

Fig. 5 Effect of arbuscular mycorrhizal (AM) symbiosis on root parasitic plant control. Under low
phosphorous conditions plants produce an increased amount of strigolactones. These signalling
molecules act as germination stimulants of root parasitic plant seeds (left). Upon mycorrhizal colo-
nization, plants reduce the production of strigolactones thus reducing parasitic plant infection, and
consequently diminishing the deleterious effect of these weeds on plant fitness and yield (right)

management through agroforestry, reduced soil disturbance, crop rotation or myc-

orrhizal inoculation would improve mycorrhizal benefits in agro-ecosystems. In
addition, these crops would take advantage of all the other well-known benefits of the
AM symbiosis such as positive effect on plant fitness and boost of plant defence
mechanisms (Fig. 5). Altogether makes AM symbiosis a suitable and promising
tool for the biological control of parasitic weeds.
So far, none of the reported approaches applied alone has led to an optimal solu-
tion against root parasitic weeds. Therefore, an integrated approach using several
strategies, including the control of seed germination, should lead to an efficient and
long-term management of this pest in agriculture.

7 Conclusion

Chemicals fertilizers and pesticides are used to prevent, mitigate or control plant
diseases. However, the environmental pollution caused by excessive use and misuse
of agrochemicals has led to public concerns about the use of these chemicals in
agriculture. Therefore, there is a need to find more environmentally friendly alterna-
tives for disease control. The key to achieve successful biological control is the
knowledge on plant interactions in an ecological context. We emphasize here the
importance of the chemical communication that occurs in the rhizosphere between
Communication in the Rhizosphere, a Target for Pest Management 129

plants and other organisms, and the potential use of this molecular dialogue as a
target to control soil-borne pathogens and pests. An interesting example is the use
of the mutualistic AM symbiosis for controlling root parasitic plant infection by
reducing the production of strigolactones by the host plant. This example illustrates
the suitability of approaches based on the knowledge of the biological system to
target. Further research will expand our knowledge on what is going on under-
ground, and the information generated will help us decipher the regulation of chem-
ical communication in the rhizosphere and may result in the development of new
biocontrol strategies against soil pests.

Acknowledgements Our research is supported by grants PERG-02-2007-224751 from the Marie

Curie program from the European Commission, AGL2009-07691 from the National R&D Plan of
the MINCIN, and The Netherlands Organisation for Scientific Research (NWO; VICI-grant to
HB). HB is also (co) financed by the Centre for BioSystems Genomics (CBSG), a part of the
Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research. JAL-R is sup-
ported by a postdoctoral contract (JAE-Doc) from the Spanish Research Council (CSIC).

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