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Chapter 11
Plant Growth-Promoting Bacteria:
Fundamentals and Exploitation
Clara Pliego, Faina Kamilova, and Ben Lugtenberg
11.1
Introduction
The rhizosphere was defined by Hiltner (1904) as “the soil compartment influenced
by the roots of growing plants.” Scientists agree that this area is not more than a few
millimeters thick. When bacteria are present on the root plane or near the plant root
we call them rhizosphere bacteria or rhizobacteria. When present on the leaf or
inside the plant tissues we speak about phyllosphere bacteria and endophytes,
respectively. When they colonize the rhizosphere they can be present on the root
surface (the “rhizoplane”) or close to the rhizosphere. The bacteria Rhizobium and
Bradyrhizobium can be present in special organs, so-called root nodules (Spaink
et al. 1998).
In comparison to bulk soil, the rhizosphere is rich in nutrients because of
rhizodeposition. Rhizodeposits consist of the total carbon transferred from the
plant root to the soil. They consist of small molecules as well as of macromolecules
including enzymes, lysates from dead cells, and mucilage. The pH of this exudate is
around 5.5 (Kamilova et al. 2006b). The result of this richness in nutrients is that the
C. Pliego
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga Consejo Superior de Investigaciones Cientı́ficas (IHSM-UMA-CSIC), Área de Genética,
Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain
and
Present address: Imperial College London. Div. of Biology, Department of Life Science, Imperial
College Road, London SW7 2AZ, UK
e-mail: [email protected], [email protected]
F. Kamilova
Koppert Biological Systems, Veilingweg 14, PO Box 155, 2650 AD Berkel en Rodenrijs,
the Netherlands
e-mail: [email protected]
B. Lugtenberg (*)
Leiden University, Institute of Biology, Sylvius Laboratory, Sylviusweg 72, PO Box 9505, 2300
RA Leiden, the Netherlands
e-mail: [email protected]
D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems,
DOI 10.1007/978-3-642-18357-7_11, # Springer-Verlag Berlin Heidelberg 2011
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bacterial concentration in the rhizosphere is 100- to 1,000-fold higher than that in
bulk soil.
Bacteria that are introduced in the rhizosphere should be able to cope with an
environment which contains other microbes (bacteria, fungi, and viruses) and
predators such as nematodes and protozoa. Also, the amount of available water
changes, especially in case of irrigation, rain, and drought. When studying the
behavior of a microbe in the rhizosphere, one should realize that exudate collected
from a gnotobiotic rhizosphere allows bacteria to grow to only a 100-fold lower cell
concentration than a laboratory medium, meaning that microbes in the rhizosphere
are often in a state of nutrient starvation.
In horticulture, plants are not only grown in soil but also in various other
substrates such as cocopeat, stonewool, perlite, and vermiculite.
11.2
Organisms in the Rhizosphere
The rhizosphere represents a highly dynamic site for interactions between roots,
pathogenic and beneficial soil microbes, invertebrates, and other competitors of
root systems. Interactions between organisms on the root may be classified as
positive, neutral, or negative associations. Positive interactions, which can contribute to plant growth promotion and biological control of plant diseases, include
symbiotic associations with epiphytes, mycorrhizal fungi, and root colonization by
biocontrol agents (BCAs). Negative interactions include competition or parasitism
among plants, pathogenesis by bacteria or fungi, and by invertebrate herbivores.
Plant–microbe interactions in the rhizosphere are responsible for a number of
intrinsic processes such as carbon sequestration, ecosystem functioning, and nutrient cycling. The composition and quantity of microbes in the soil influence the
ability of a plant to obtain nitrogen and other nutrients. On the other hand, plants
can influence these net ecosystems changes through release of secondary metabolites that attract or inhibit the growth of specific microorganisms (Lugtenberg and
Bloemberg 2004; Vivanco et al. 2004). In addition, the rhizosphere is subject to
dramatic changes on a short temporal scale, which can be caused by fluctuations in
salt concentration, pH, osmotic potential, water potential, and soil particle structure.
Over longer temporal scales, the rhizosphere can change due to root growth and
interactions with other soil biota.
Various bacterial species have adapted to the ever-changing conditions of the
rhizosphere and are capable of starting root colonization by multiplication and
eventually by forming microcolonies on different parts of the roots, from tip to
elongation zone. Such microcolonies eventually grow on roots to form mature
biofilms (Bloemberg and Lugtenberg 2004; Rudrappa et al. 2008). Nonpathogenic
plant growth-promoting rhizobacteria (PGPR) associated with plant root surfaces
are known to contribute toward increases in plant quality and yield by mechanisms
such as improved mineral uptake, phytohormone production (Biswas et al. 2000;
Ryu et al. 2003; Ryu et al. 2004; Fujishige et al. 2006), competitive suppression of
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pathogens by production of antibiotics (Chin-A-Woeng et al. 1998; Keel et al.
1992; Haas and Défago 2005), and induction of secondary metabolite-mediated
systemic resistance (Ryu et al. 2004; Van Loon 2007). Besides, bacterial biofilms
established on plant roots can protect the colonization sites and act as a sink for the
nutrients in the rhizosphere, thereby reducing the availability of nutritional elements in root exudates required for pathogen stimulation or subsequent root colonization (Weller and Thomashow 1994; Kamilova et al. 2005).
In addition to the interaction with the plant, beneficial microorganisms interact
and compete with the endogenous microflora, consisting of other bacteria, fungi,
and/or mycorrhizal fungi. It is the dynamic nature of the rhizosphere that makes it
an interesting setting for the interactions that lead to plant growth promotion and
biocontrol of the disease. Significant biological control most generally arises from
manipulating mutualisms between microbes and their plant hosts or from manipulating antagonisms between beneficial microbes and pathogens. To do this successfully, we need to know the players and understand their interactions with each
other and with the growth substrate. Moreover, the effect of abiotic factors should
be taken into account. The analysis of these interactions requires a strong interdisciplinary approach and an extensive set of tools.
11.3
Nutrients in the Rhizosphere
Compounds released by the plant root vary in relative and absolute amounts. The
composition is influenced by the plant species, plant cultivar and age, and environmental properties such as the presence of microbes and some of their products, the
growth substrate, and the levels of physical, biological, and chemical stresses. It
should be realized that an exudate composition usually reflects the net result of
secretion, conversion by enzymes, and uptake by microbes and the plant root. An
excellent monograph on the rhizosphere was published recently (Pinton et al. 2007).
The bulk of root products are carbon compounds derived from products of photosynthesis. An excellent overview of exudate compounds is written by Uren (2007).
Organic compounds released by the root include organic acids, sugars and polysaccharides, amino acids, fatty acids, sterols, vitamins, enzymes, and nucleotides.
Moreover, compounds as diverse as auxins, ethanol, flavonoids, and the osmoprotectant glycinebetaine are secreted. In many cases, compounds are only detected because
one screens for their presence. Exudate compounds can also be detected accidentally.
Kuiper et al. (2001a) studied a competitive colonization mutant and the identification
of the knocked out gene suggested that putrescine could be an exudate compound.
Subsequent tests confirmed this notion. The rhizosphere is also an important place for
microbe–plant and microbe–microbe signaling (Perry et al. 2007).
Rhizobacteria (Kamilova et al. 2006b), fungi (Meharg and Killham 1995), and
rhizobacterial products such as phenazine, 2,4 diacetylphloroglucinol and zearalenone (Phillips et al. 2004), as well as the growth substrate (Kamilova et al. 2006a;
Lipton et al. 1987), can influence the exudate composition.
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Extensive studies have been carried out on the exudate composition of gnotobiotically grown tomato, cucumber, and sweet pepper. For all three crops, the
organic acid fraction was larger than the carbohydrate fraction which, in turn,
was larger than the amino acid fraction (Kamilova et al. 2006a). The observation
that a mutant of Pseudomonas unable to utilize organic acids is a poor colonizer
(De Weert et al. 2007) but a mutant in sugar utilization can colonize the tomato root
normally (Lugtenberg et al. 2001) is consistent with the notion that organic acids
are a major carbon source.
The presence of a disease-causing amount of the pathogenic fungus Fusarium
oxysporum f. sp. radicis-lycopersici in the gnotobiotic tomato system resulted in a
decrease of the amount of citric acid and an increase in the amount of succinic acid
in such a way that the total amount of organic acid was not influenced. In contrast,
when the Pseudomonas fluorescens biocontrol strain WCS365 was added to the
gnotobiotic tomato system in amounts sufficient to cause biocontrol, a strong
increase in the total amount of organic acid, especially of citric acid, but a dramatic
decrease in the amount of another organic acid, succinic acid, was found. When
both microbes were present under biocontrol conditions, the amounts of succinic
acid decreased but the amount of citric acid was similar to that of the untreated
control. Under all three conditions, the amounts of sugars were reduced by around
50% (Kamilova et al. 2006b).
In contrast to the three dicots cucumber, sweet pepper, and tomato, the monocot
grass produces equal amounts of organic acids and sugars in its root exudate (Kuiper
et al. 2002). Whether this difference reflects a fundamental difference in exudate
composition between monocots and dicots remains to be established.
Although root exudates play a crucial role in plant growth and health, studies on
composition, the influence of environmental factors on composition, and function
of root exudates have hardly been performed in the past 4 decades. Such studies
should be considered as a challenge for future research.
11.4
Root Colonization
Root colonization is crucial for the application of beneficial bacteria. Recently,
technologies for following root colonization have been developed and traits and
genes involved in colonization have been identified.
11.4.1 Recently Developed Technologies for Studying
Interactions in the Rhizosphere
Until recently, most research on biological control has focused on the interactions
between two trophic levels: the BCA and the pathogen. However, multitrophic
interactions among organisms belonging to three or more trophic levels are increasingly gaining the attention of microbial ecologists and plant pathologists, driven by
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the need for improving biological control during the management of crop diseases.
As we have mentioned previously, successful biocontrol operates at the population
level, not at the level of an individual organism. Consequently, a detailed understanding of species interactions across at least three levels, the plant, the pathogen,
and the BCA, seems to be necessary (Pliego et al. 2008). During the last years, the
development of technologies such as the bioreporter technology has dramatically
influenced our knowledge of biocontrol in the rhizosphere. Bioreporters can be
classified into three categories (Lugtenberg and Leveau 2007). One class of bioreporters are those that allow direct visualization of individual organisms in vivo.
The most common reporter used for this purpose is the green fluorescent protein
(GFP), which does not depend on cofactors or additional substrates for activity. In
combination with a constitutive promoter, the gfp gene is expressed independently
of environmental factors and accumulation of its product renders the cells green
fluorescent and detectable by fluorescence microscopy or confocal laser-scanning
microscopy (CLSM). This has been exploited to study the direct visualization of the
interactions established among various organisms, i.e., BCAs and pathogenic fungi.
The second class allows monitoring the general well-being or activity of BCAs in
the rhizosphere. Some groups have used the lux genes, which only results in
bioluminescence when the cells are metabolically active, to show that the metabolic
activity of Pseudomonas inoculants is higher in rhizosphere soil than in bulk soil
and that metabolic activity decreases over time after introduction of the inoculated
BCA (Porteous et al. 2000). The third group of bioreporters is aimed at providing
information on specific activities of BCAs in the rhizosphere. The expression of the
reporter gene is driven by promoter sequences that are selected based on their
biological function. Besides gfp and lux, other reporter genes can be used for this,
including lacZ, xylE, gusA, and inaZ (Loper and Lindow 1997).
Finally, Transposon-Based-Strategies (TSBs) and the development of functional
genomic techniques such as In Vivo Expression Technology (IVET), Signature
Tagged Mutagenesis (STM), and DNA microarrays allow the identification of
microbial genes potentially involved in biocontrol. Application of these techniques
is largely increasing our knowledge of the molecular mechanisms involved in the
interactions established among different trophic levels.
11.4.2 Autofluorescent Proteins (AFPs)
Microscopic visualization of AFP-tagged BCAs in their natural environment during
their interaction with plants and/or with target pathogens provides crucial information about their functioning and about the possible successful application of commercial inoculants. The GFP is the most widely studied AFP but the number of other
proteins and variants with different fluorescence characteristics has increased. This
also applies for the development of improved variants of existing AFPs. Important
GFP derivatives are Cyan Fluorescent Protein (CFP) and Yellow Fluorescent
Protein (YFP) (Yang et al. 1998; Tsien 1998; Matus 1999; Ellenberg et al. 1999).
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A wide range of autofluorescent proteins with useful information is available on the
Web site of the Clontech company (http://www.clontech.com). Taking into account
that genes encoding AFPs have to be transformed into the organism of interest, the
use of these markers is dependent on whether appropriate vectors and transformation
protocols are available for a specific species or strain. In bacteria, AFP genes are
usually delivered using plasmids or transposons (Bloemberg 2007). The advantage
of a plasmid is that it can be present in multiple copies, which can improve the level
of AFP molecules and does not disrupt hosts genes by chromosomal integration
(Bloemberg 2007).
In recent years, AFP technology in combination with CLSM has become an
important tool for the analysis of processes such as microbe–plant interactions,
biosensor design, biofilm formation, and horizontal gene transfer (Larrainzar et al.
2005). Besides, the ability to use AFPs with different fluorescent spectra allows the
simultaneous visualization of different species and populations at the same time.
The use of red fluorescent protein (RFP), isolated from Discosoma striata, in
combination with eGFP, is very suitable as the excitation and emission spectra of
these proteins are well separated (Matz et al. 1999). Because of the functional
limitations of DsRed such as slow maturation and low photostability, new and
improved variants have recently been produced, of which mCherry is one of the
best (Shaner et al. 2004). For example, Lagendijk et al. (2010) were able to
distinguish simultaneously in the tomato rhizosphere the presence of mixed microcolonies originated from two different populations of P. putida PCL1445 cells
tagged with EGFP and mCherry.
Most of the studies using AFP as markers for BCAs have been directed to their
localization in the rhizosphere. These studies have shown that individual bacteria
grow out to microcolonies and form biofilms, usually at junctions between epidermal root cells and at sites where side roots emerge. These are supposed to be
microhabitats with enhanced exudation, humidity, and mucigel (Lugtenberg and
Bloemberg 2004). Studies using mixed populations of two Pseudomonas species,
P. chlororaphis PCL1391 and P. fluorescens WCS365, revealed that mixed colonies were formed and are present mostly on the upper part of the root and that
P. fluorescens WCS365 was found preferentially on root hairs (Lagopodi et al.
2002). These studies revealed that different bacteria can have a preference for
colonizing different parts of the roots. The most significant studies related with
the use of AFPs to study the interaction of Pseudomonas spp. with plant roots are
given in Table 11.1.
Epifluorescence studies also showed the close interaction established between
rhizosphere fungi and bacteria. Using this technique, bacterial cells have been
shown to attach and colonize the hyphae of beneficial and phytopathogenic fungi.
Colonization of fungal hyphae by antagonistic bacteria has also been postulated to
enhance biocontrol in combination with the bacterial production of antifungal
metabolites such as antibiotics, chitinases, or proteases (Hogan and Kolter
2002; Bolwerk et al. 2003; Lagopodi et al. 2002). For example, the biocontrol
strains Collimonas fungivorans, P. fluorescens WCS365, and P. chlororaphis
PCL1391 have not only been shown to be excellent root colonizers but appeared
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Table 11.1 Microscopic visualization of AFP-tagged Pseudomonas strains in the plant rhizospherea
Pseudomonas strain
System
P. fluorescens WCS365 Tomato
Localization
Promoter::
gfp-version
(Plac: gfp)
P. chlororaphis
Lotus
Localization
(PpsbA::gfp)
P. fluorescens
DR54-BN14
P. fluorescens WC5365
Barley
Localization and
activity
Localization
P. fluorescens SBW25
Wheat
(PA1/03/04:
gfpmut3b)
(Plac::rfp; Plac::
egfp; Plac::ecfp;
Plac::eyfp)
(PpsbA-gfp luxAB)
P. putida
Barley
P. fluorescens F113
Alfalfa
Tomato
P. fluorescens WCS365 Tomato
and P. chlororaphis
PCL1391
P. brassicacearum
Arabidopsis
thaliana
Analyzed trait
Metabolic activity
and localization
Activity
(PrrnBP1::gfp
Localization
[AGA])
(PA1/03/04:gfpmut3
Localization
(Plac:gfpS65T)
Localization
(Plac::rfp)
Distribution
(PA1/03/04:
RBSIIgfpmut3)
(PA1/03/04:RBSIIdsred)
(PA1/03/04:gfpmut3)
(Plac:gfp)
P. putida
P. fluorescens 32
Tomato
Wheat
Localization
Localization
P. fluorescens CHA0
Set of plant
species
Avocado
Gene expression
Tomato
Localization
P. pseudoalcaligenes
and P. alcaligenes
P. putida PCL1445
Distribution
(PphlA-gfp3)
(PprnA-gfp3)
(PA1/03/04:gfp)
(Ptac:gfp; Ptac:
mcherry)
Reference
Bloemberg et al.
(1997)
Tombolini et al.
(1997)
Normander et al.
(1999)
Bloemberg et al.
(2000)
Unge and Jansson
(2001)
Ramos et al. (2000);
Boldt et al. (2004)
Villacieros et al.
(2003)
Bolwerk et al. (2003)
Achouak et al. (2004)
G€
otz et al. 2006
Van Bruggen et al.
(2007)
De Werra et al. (2008)
Pliego et al. (2008)
Lagendijk et al. (2010)
a
PpsbA, Plac, Ptac, PA1/03/04 (a Plac-derivative) are constitutive promoters; PrrnBP1 is a ribosomal
promoter; PphlA contains the promoter region of the genes encoding the antifungal metabolite
2,4-diacetylphloroglucinol; PprnA contains the promoter region of the genes encoding the antifungal metabolite pyrrolnitrin; gfp, egfp, gfpmut3b, gfpmut3, and gfpS65T are genes encoding stable
versions of the GFP protein; gfp[AGA] is a gene encoding an unstable version of the GFP protein;
dsred and rfp are genes encoding derivatives of the GFP protein emitting red fluorescence; yfp is a
gene encoding a yellow fluorescent protein; luxAB genes encode luciferase from Vibrio fischeri
also to be able to colonize the hyphae of the phytopathogenic fungus F. oxysporum
f.sp. radicis lycopersici (Forl) (Lagopodi et al. 2002; Bolwerk et al. 2003;
Kamilova et al. 2007). Furthermore, abundant colonization of hyphae of the fungus
Rosellina necatrix, the causal agent of avocado white root rot, was observed for the
biocontrol strain P. pseudoalcaligenes AVO110 (Pliego et al. 2008). Moreover, the
latter strain also colonized profusely pyriform swellings, characteristic structures of
R. necatrix hyphae that seem to be involved in chlamydospore formation (PérezJiménez et al. 2003).
Since fungi are frequently part of the endogenous microflora and can be the
direct target for the PGPR effect as in case of biological control, it is also of great
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relevance to tag fungi and study their interaction with the PGPR (Bloemberg 2007).
Genetic transformation of filamentous fungi is usually more difficult and less
developed than transformation of bacterial cells. However, GFP expression vectors
have already been developed for all major classes of filamentous fungi: basidiomycetes, ascomycetes, and oomycetes. The vector to be used depends on the fungus
to be transformed. Transformation of filamentous fungi has traditionally been
performed using protoplasts, e.g., F. oxysporum f.sp. radicis lycopersici has been
tagged with different autofluorescent proteins by co-transformation of two plasmids, one of which contained a hygromycin resistance gene and the second an afp
gene (Lagopodi et al. 2002; Bolwerk et al. 2003). Until now, several root-infecting
fungal pathogens have been tagged with GFP to study developmental processes
occurring during root infection, for example, Magnaporte grisea (Sesma and
Osbourn 2004), Forl (Lagopodi et al. 2002), Fusarium verticillioides (Oren et al.
2003), and Rosellinia necatrix (Pliego et al. 2009).
These studies have made significant contributions to the understanding of
how BCAs function during their beneficial interactions with plants. For example,
Lagopodi et al. (2002) observed that the biocontrol bacteria P. fluorescens WCS365
and P. chlororaphis PCL1391, applied to seedlings, and the pathogenic fungus
Forl, applied in the soil, occupy the same sites on the tomato root mostly in the
junctions between cells. Furthermore, the observation that the biocontrol strain
occupies these sites faster that the pathogenic fungus presumably underlies a crucial
aspect of the bacterium’s biocontrol ability (Bolwerk et al. 2003). More recently,
CLSM studies revealed that the biocontrol strain P. pseudoalcaligenes AVO110 is
able to control the avocado white root rot disease caused by R. necatrix by
colonizing profusely the same sites as used by the pathogen to penetrate the root
such as root wounds and intercellular spaces (Pliego et al. 2008).
11.4.3 Colonization Genes and Traits
One of the major challenges for researchers is to understand the role of bacteria in
their natural environment. One way to do this is to identify genes that are specifically expressed in natural surroundings. As mentioned earlier, efficient protection
of plant roots by BCAs firstly requires sufficient and competitive colonization of the
rhizosphere. In this regard, most studies have been directed to the identification of
bacterial genes involved in rhizosphere colonization. Two different approaches
were used to identify traits involved in root colonization and rhizosphere competence for the model strain P. fluorescens WCS365, one of the best colonizers among
biocontrol strains (Lugtenberg et al. 2001). These studies were facilitated by the
development of a gnotobiotic system developed by Simons et al. (1996) which
offers a sterile environment for comparing the wild type with various mutants.
The first approach deals with bacterial genes predicted to have an effect on
colonization. These genes can be disrupted by directed mutagenesis using homologous recombination. Prior to studying their root colonizing abilities, growth rate
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and other differences between mutant and wild type should be established. Using
this approach, the importance of, for example, motility and chemotaxis (Lugtenberg
and Bloemberg 2004) for root colonization was demonstrated. In addition, using
cheA mutants of various P. fluorescens strains, De Weert et al. (2002) showed that
the presence of flagella is not sufficient for competitive colonization but that
chemotaxis in addition is essential. Motility is required for the chemotactic
response of microbes toward exudates compounds as a first step in the process of
root colonization (De Weert et al. 2007). Major chemoattractants in tomato root
exudates for P. fluorescens WCS365 are dicarboxylic acids and amino acids. Based
on concentrations estimated to be present in the tomato rhizosphere (Simons et al.
1997; Kravchenko et al. 2003), malic acid and citric acid were concluded to be the
major chemoattractants.
As a second approach, random transposon mutagenesis can be performed using
transposons such as Tn5lacZ (Lam et al. 1990) or Tn5luxAB (Wolk et al. 1991).
Selected mutants can be studied individually for impaired root colonization
(Simons et al. 1996). Applying of the transposon approach resulted in the discovery
of relevant traits for rhizosphere colonization. An overview of these traits is
presented in Table 11.2. Reviews of most of these traits can be found in Lugtenberg
and Dekkers (1999), Lugtenberg et al. (2001), De Weert et al. (2007), and Lugtenberg
and Kamilova (2009).
Functional genomic techniques allow the simultaneous in vivo examination of
the expression of many genes. These techniques can be divided into three broad
Table 11.2 Functions of Pseudomonas genes required for root colonization
Function
Gene
References
Motility
Involved in biosynthesis of flagella
De Weger et al. (1987)
Pilus retraction
pilT
Camacho Carvajal
(2001)
Chemotaxis
cheA
De Weert et al. (2002)
Cell wall structure
rhs
De Weert et al. (2007)
Synthesis of O-antigen
Not identified
Dekkers et al. (1998)
of LPS
Outer membrane integrity
Homolog to hrtB
Dekkers et al. (1998)
Permeability of outer
colR/colS required for expression
De Weert et al. (2006)
membrane
of wapQ
Increased pyrimidine
pyrR
Camacho Carvajal
biosynthesis
(2001)
Utilization of exudate
Homolog of mqo encoding malate
Lugtenberg et al. (2001)
compounds
dehidrogenase
De Weert and
Homolog of cis-aconitate hydratase
Bloemberg (2007)
Downregulation of
Binding site for regulator protein
Kuiper et al. (2001a)
putrescine uptake
Phase variation
xerC/sss
Dekkers et al. (1998)
Proton motive force
nuo operon
Camacho Carvajal et al.
(2002)
Secretion pathways
secB, homolog of hrcC, hrcD encoding
De Weert et al. (2007)
components of the TTSS
Adaptation to certain
Homolog of mutY
De Weert et al. (2004a)
environments
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classes (1) IVET, (2) STM, and (3) transcriptomics, i.e., global gene expression
analysis using DNA microarrays. IVET technology is a promoter-trap strategy
designed to identify genes whose expression is induced in a specific environment,
typically that encountered in a host. This technique was, for example, used to
identify genes of P. fluorescens specifically expressed within the sugar beet rhizosphere (Rainey 1999; Gal et al. 2003) and to analyze gene expression in P. putida
during colonization of the maize rhizosphere (Ramos-González et al. 2005).
Rhizosphere-induced fusions identified genes with probable functions in nutrient
scavenging, chemotaxis and motility, intracellular metabolism, regulation, secretion, cell envelope structure, nucleic acid metabolism and stress. One of the most
important findings was the discovery of the hrC gene, encoding a component of the
Type Three Secretion System (TTSS) of P. fluorescens (Rainey 1999), suggesting
that the TTSS has functional significance in both pathogens and PGPR. Although
TTSS in P. fluorescens was demonstrated to be functional, a hrcC mutation did not
affect root colonization capacity (Preston et al. 2001). On the other hand, De Weert
et al. (2007) compared the competitive root-colonizing abilities of both hrcD and
hrcD genes of the TTSS of P. fluorescens SBW25. It appeared that both mutants
were impaired in competitive tomato root tip colonization but were not impaired
when analyzed alone on the root. They hypothesized that the mutants are using the
TTSS for either involvement in attachment to seed and/or root or for feeding on the
root surface cells by means of the TTSS. Because no difference was found in
attachment to seeds or roots, in competition with the wild type, they suggested
that the TTSS is not only used to inject proteins into plant cells but also to suck
nutrients from the plant cell (De Weert et al. 2007). These results support the
finding that biocontrol activity of P. putida against Pythium ultimum on cucumber
was lower that that of the wild type when the hrcC gene was mutated (Rezzonico
et al. 2004).
Interestingly, application of IVET technology also led to the identification of the
P. fluorescens wssE gene, being part of the wss operon involved in the synthesis of
acetylated cellulose polymers (Rainey and Preston, 2000; Gal et al. 2003). The
wssE gene encodes a cellulose synthase subunit. Acetylated cellulose polymers are
known to play a role in colony development and bacterial cell–cell contact, which
are known to be involved in the initial steps of biofilm development (Spiers et al.
2003). Mutational analysis of the wss operon has shown that it contributes to
ecological fitness in the rhizosphere. Besides, an increased expression of oxidative
stress genes (glutathione peroxidases and a paraquat inducible protein) was
observed in P. fluorescens for protection against oxidative stress encountered in
the sugar beet rhizosphere (Gal et al. 2003).
In the functional genomic technique STM, the use of DNA signature tags
facilitate functional screens by identifying mutants in mixed populations that
have a reduced or increased adaptation to a particular environment. Besides,
STM allows the identification of fitness or competition mutants, usually missed
by standard mutagenesis approaches, which do not cause a total phenotype knockout. This technique was recently applied in Burkholderia vietnamiensis strain G4 to
identify genetic determinants involved in colonization of the pea rhizosphere and in
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phenol degradation. Seventy mutants involved in rhizosphere colonization were
mapped to genes with the following putative functions: amino acid biosynthesis
(36%), general metabolism (26%), transport and stress (2.8%), regulatory genes
(5.7%), and hypothetical proteins (13%) (O’Sullivan et al. 2007). The majority of
the mutants were associated with amino acid biosynthesis and metabolism. Amino
acids are known to be present in root exudates but at limiting concentrations and not
readily accessible (Lugtenberg et al. 2001). Several amino acid auxotrophs were
also found for P. fluorescens WCS365 among competitive colonization mutants.
Even when tested alone, these mutants were unable to colonize the root tip
(Lugtenberg and Bloemberg 2004).
One of the most interesting discoveries mediated by the rhizosphere-STM screen
was the identification of three mutants, inactivated within a single virulenceassociated autotransporter adhesion gene (O’Sullivan et al. 2007). This mutation
consistently produced a hypercolonization phenotype. They suggests that this
massive surface-exposed protein (229–368 Da) masks part of the surface adhesins
that are actually responsible for plant root cell binding, hence these protein adhesins
would be more freely available for binding to plant cells in these three mutants.
Alternatively, this adhesion could also play a role in the plant host response, in a
way analogous to the way the known immunogenicity YadA homologs act in
animal hosts (Cotter et al. 2005). Therefore, it may be possible that the plant is
more tolerant to the adhesin mutants and allows them to colonize in greater
numbers (O’Sullivan et al. 2007).
Using a transposon mutant library of the efficient colonizer P. fluorescens
WCS365 in combination with the enrichment procedure developed by Kuiper
et al. (2001a), De Weert and co-workers (2004a) observed that competitive root
tip colonization can be enhanced. The best colonizing mutant was shown to be
mutated in a mutY homolog. This gene is involved in the repair of A:G mismatches
caused by spontaneous oxidation of guanine. Since these mutants are defective in
repairing their mismatches, it is hypothesized that such cells harbor an increased
number of mutations and that some of these mutants, with a beneficial combination
of mutations, can adapt faster to the environment of the root system.
In order to investigate how Pseudomonas populations readjust their genetic
program upon establishment of a mutualistic interaction with plants, Matilla et al.
(2007) performed a genome-wide analysis of gene expression using microarrays of
the root-colonizing bacterium P. putida KT2440. Genes involved in amino acid
uptake and metabolism of aromatic compounds were found to be preferentially
expressed in the rhizosphere (22%). It was also found that bacterial efflux pumps
and enzymes related with glutathione metabolism were induced in the rhizosphere,
indicating again that adaptation to adverse conditions and to oxidative stress is
crucial for bacterial life in this environment. A gene encoding a protein containing
GGDEF/EAL domains was identified among the induced genes. These domains are
involved in c-di-GMP metabolism. Numerous studies have shown that c-di-GMP
regulates biofilm formation and motility. Moreover, c-di-GMP also activates the
production of extracellular polysaccharides (EPS), specifically those that serve
as an extracellular matrix for formation and support of biofilm architecture
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(Tamayo et al. 2007). Results shown by Matilla et al. (2007) suggest a role for the
turnover of c-di-GMP in root colonization. Besides, genes encoding flagellar
proteins and chemotaxis proteins were also upregulated in the interaction of
P. putida KT2440 with maize roots, confirming a role of chemotaxis in root
colonization.
11.5
Bacteria Which Can Promote Plant Growth Directly
Some beneficial bacteria can promote plant growth in the absence of a pathogen.
They will be discussed here.
11.5.1 Introduction
Some rhizobacteria are plant beneficial and can promote plant growth directly, e.g., by
providing the plant with nutrients or growth hormones, whereas others can promote
plant growth indirectly, e.g., by decreasing the growth of pathogens. Direct plant
growth promotion will be discussed in this section whereas indirect plant growth
promotion will be treated in the next section. Direct plant growth promoters can be
divided into four classes: biofertilizers, rhizoremediators, phytostimulators or plant
growth regulators, and stress controllers.
11.5.2 Biofertilizers
Biofertilizers are best known for their ability to provide the plant root with nutrients
such as nitrogen, phosphorous, and iron.
Biological nitrogen fixation is an extremely energy intensive process. The
reduction of one molecule of N2 catalyzed by the nitrogenase enzyme complex
requires between 16 and 42 molecules of ATP. It is therefore not surprising that
N2-fixing bacteria only reduce N2 when they are starved for nitrogen (De Bruijn
et al. 1990). Rhizobium and Bradyrhizobium bacteria can induce special organs,
so-called root nodules on the roots of leguminous plants in which they, in the form
of modified bacteria called bacteroids, fix atmospheric nitrogen and convert it into
ammonia which can be utilized by the plant as a nitrogen source. In return, the plant
provides the bacterium with a carbon source, probably malate. Both rhizobia are
economically important. Bradyrhizobium is important in agriculture as it can
nodulate soybean. Rhizobium can nodulate crops such as pea, peanut, and alfalfa.
Egamberdieva et al. (2010) recently showed that co-inoculation of fodder galega
with Rhizobium and biocontrol pseudomonads improves shoot and root dry matter
of the plant. One of these strains, the cellulose-producing P. trivialis strain 3Re27,
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
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significantly increased nodule numbers and nitrogen content of the co-inoculated
plant (Egamberdieva et al. 2010). The authors coined the term “rhizobium helper
bacteria” for this biocontrol strain.
Klebsiella pneumonia and Azospirillum are free-living nitrogen-fixing rhizosphere
bacteria. In the past, the plant growth-promoting properties of Azospirillum were
thought to be due to its N2-fixing property, but recent developments show that this
property is mainly due to its ability to produce the root architecture influencing
hormone auxin. Therefore, Azospirillum will be discussed under “Phytostimulators.”
In some soils, phosphorous is the limiting factor for plant growth. Some bacteria
are able to solubilize bound phosphorous from organic or inorganic molecules,
thereby making it available for the plant (Lipton et al. 1987; Vassilev et al. 2006).
Production of organic acids such as gluconic acid is a major factor in the release of
phosphorous from mineral phosphate (Rodrı́guez et al. 2006).
All organisms need iron ions for their metabolism. Iron in the form of ferric
hydroxide is an abundant element on the earth crust but it is insoluble and therefore
not suitable for uptake by living organisms. The concentration of Fe3+, the form of
iron ions available for living organisms, is only 1018 M. Therefore, bacteria growing
under low Fe3þ concentrations produce a variety of siderophores, which are Fe3þ
chelators able to bind this ion with high affinity. Subsequently, the Fe3þ-siderophore
complex is bound to specific iron starvation induced bacterial cell surface receptors
and the Fe3þ ion is transported into the cell’s interior, where it, in the form of Fe2þ, is
involved in metabolic processes (Neilands 1981; Leong 1986).
Competition for ferric iron ions is a well-documented example of competition of
biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986; Lugtenberg
and Bloemberg 2004). In the rhizosphere, there is often a limitation of solubilized
Fe3þ which, in many microorganisms, is solved by the production of siderophores,
i.e., Fe3þ-binding ligands, which have a high affinity to sequester iron from the
microenvironment. The relevance of siderophore production as a mechanism of
biological control of Erwinia carotovora by P. fluorescens strains was first described
by Kloepper et al. (1980). As with antibiotics, mutants not producing siderophores
such as pyoverdine showed a reduced capacity to suppress different plant pathogens
(Keel et al. 1989; Loper and Buyer 1991; Duijff et al. 1994). This phenomenon is not
only a form of plant fertilization but also of biocontrol (see Sect. 6.2.3).
11.5.3 Rhizoremediators
Pollution of soil and water is an enormous problem worldwide. In the USA alone,
the costs of restoration of all contaminated sites are estimated at 1.7 trillion USDs.
The major approaches to solve these problems are incineration and removal of land.
However, these approaches are not safe. Incineration results in air pollution and
leaches from landfills can reach ground water and drinking water wells. Microbes at
a site that is being polluted adapt to this situation and start degrading the pollutants.
However, the degradation rate is very low. Scientists have isolated the microbes
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responsible for this process. For most contaminants it is possible to find microbes
which can degrade them. The application of such microbes has been tested for
decades. However, strains which were successful in the laboratory failed in soil.
Due to lack of nutrients they go in a starvation mode soon after application and stop
degrading the pollutants. Apparently, they are unable to multiply on the pollutants
as their only carbon source. In order to degrade the pollutant molecules they have to
co-metabolize it together with a regular C source (Cases and De Lorenzo 2005).
With the notion, that it is possible to find bacteria able to solve almost any
problem, Kuiper et al. (2001b) selected bacteria which combine two properties,
namely (1) to utilize root exudate – the best carbon source available in soil and (2)
are also able to degrade pollutants. Grass was used as the exudate source because
the roots of some grasses can grow as deep as 5 m into the soil. The polyaromatic
hydrocarbon compound naphthalene was chosen as the model pollutant. Starting
with the crude rhizosphere of the roots, presumably containing a mixture of
hundreds of thousands different rhizobacteria, the mixture was grown first on
naphthalene as the only carbon source. The naphthalene users were subsequently
coated on grass seeds, which were allowed to grow until the roots were 10 cm in
length. Subsequently, the bacteria residing on the last centimeter of the root tip,
i.e., the best root colonizers, were collected. This cycle was repeated twice (SeeFig. 11.1 for the principle of the enrichment procedure). In this way, strain P. putida
PCL 1444 was isolated (Kuiper et al. 2001b). This strain combines the following
properties (1) stable degradation of naphthalene; (2) efficient utilization of grass
root exudate; (3) protection of coated grass seeds from poisoning by naphthalene,
resulting in healthy plants. Mutants unable to degrade naphthalene are inactive in
this respect; (4) roots are able to move bacteria through layers through which
bacteria normally cannot penetrate (Kuiper et al. 2001b; Kuiper et al. 2002; Kuiper
et al. 2004).
In addition to rhizosphere bacteria, endophytes are often used in rhizoremediation. Reports on the ability of several endophytic bacteria to degrade some pollutants (i.e., explosives, herbicides, or hydrocarbons) have been published (Van Aken
et al. 2004; Germaine et al. 2006; Phillips et al. 2008; Segura et al., 2009). Also,
endophytic bacteria resistant to high concentrations of heavy metals, benzene,
toluene, ethylbenzene and xylenes (BTEX), trichloroethylene (TCE), or polyaromatic hydrocarbons (PAHs) have been identified (Moore et al. 2006; Doty 2008).
11.5.4 Phytostimulators/Plant Growth Promoters
The plant can produce phytohormones, i.e., compounds which can regulate plant
growth and development. There are five classes of plant hormones, namely auxins,
cytokinins, gibberellins (GAs), abscisic acid (ABA), and ethylene (ET). See
Fig. 11.2 for structures. They are usually effective at concentrations lower than
1 mM (Garcı́a de Salome et al. 2006). In addition to plants, many rhizosphere
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
309
Fig. 11.1 Enrichment of bacteria which compete efficiently for nutrients and niches. The principle of the enrichment procedure was published by Kuiper et al. (2001b). Starting from a seed on
which a crude mixture of rhizosphere bacteria has been applied, enhanced competitors are selected
by repeatedly selecting those bacteria which reach the root tip first after application to a sterile
seed. Strains isolated from the root tip after three cycles are subsequently tested on antibiotic
activities toward pathogens, which results in (1) strains which are not antagonistic and easy to
register as a product (Kamilova et al. 2005) or (2) in strains which have at least two mechanisms of
biocontrol, namely CNN and antibiosis (Pliego et al. 2007; Pliego et al. 2008)
bacteria can produce plant growth regulators. The first three classes of plant
hormones will be discussed under “phytostimulators/plant growth promoters”
whereas the last two classes will be discussed under “stress controllers.” For details
on hormones produced by plants and rhizosphere bacteria, the reader is referred to
excellent reviews by Spaepen et al. (2009) and Garcı́a de Salome et al. (2006).
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C. Pliego et al.
O
COOH
C
NH2
OH
N
N
N
N
N
N
CH3
Phenazine-1-carboxylate
O
Phenazine-1-carboxamide
Pyocyanin
O
OH
NH
CH3
H3C
HO
OH
Cl
HO
CL
O
OH
NO2
HN
Cl
Cl
2,4-Diacetylphloroglucinol
Pyrrolnitrin
Pyoluteorin
CH3
CH2
CH2
CH2
CH2
CH2
NH2
O
HO
O
H
N
O
HO
N
H
N
H
N
OH
O
O
O
HN
NH
O
OH
H
H
CH2
O
HN
O
CH2
N
H
Viscosinamide
OH
O
N
H
OH
O
CH3
2-hexyl-5- propyl resorcinol
O
O
N
H
H
AHL structure
Fig. 11.2 (Continued)
O
H
C
N
Hydrogen cyanide
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
O
311
OH
OH
CO
H
CH2
HO
O
COOH
N
H
CH3
Gibberellins (GA3)
Auxins (IAA)
HO
H3C
NH
H3C
N
N
N
CH3
CH3
OH
N
H
CH3
O
Zeatin
O
OH
Abscisic acid
OH
O
OH
OH
OH
H2C
CH2
Ethylene
OH
OH
D-gluconic acid
Fig. 11.2 Structures of important metabolites which play a role in the interaction between plantbeneficial bacteria and plants
11.5.4.1
Auxins
Auxins are involved in several processes such as establishment of polarity in
embryogenesis, cell elongation, vascular differentiation, floral and fruit development, lateral root formation, and determination of root and shoot architecture.
Indole-3-acetic acid (IAA) (Fig. 11.2) is the most abundant member of the auxin
family. It has been estimated that as much as 80% of the rhizosphere bacteria can
synthesize IAA (Khalid et al. 2004; Patten and Glick 1996). Rhizosphere bacteria
use several different pathways for IAA biosynthesis. Most of them use tryptophan
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secreted in the rhizosphere as a precursor (Costacurta and Vanderleyden 1995;
Spaepen et al. 2009). Indeed, Kamilova et al. (2006a) observed that P. fluorescens
biocontrol strain WCS365, which can produce IAA in the presence of tryptophan, is
able to stimulate root growth of radish, a plant which secretes high amounts of
tryptophane, but not of tomato, sweet pepper, or cucumber, plants which secrete at
least tenfold less tryptophan in the growth medium.
Azospirillum brasilense is an N2 fixer, which promotes plant growth by increasing its root surface through shortening the root length and enhancing root hair
formation. It has been thought for a long time that its plant growth-promoting
ability was based on N2 fixation. However, the present notion is that auxin production is the major factor responsible for its root changes and therefore for its plant
growth-promoting properties. This notion is based on the following observations
(1) Dobbelaere et al. (1999) showed that the effect of the wild-type strain on the root
can be mimicked by the addition of pure auxin. (2) A mutant strain strongly reduced
in IAA production did not induce the root changes. (3) A strain constitutive for IAA
production showed the same effect on the root changes as the wild-type strain but
already at lower bacterial cell concentrations (Spaepen et al. 2008). Interestingly,
when the amount of root exudate becomes limiting for growth, A. brasilense cells
increase their IAA production, thereby triggering lateral root and root hair formation, which results in more exudation and therefore in further bacterial growth.
In this way, a regulatory loop is created which connects plant root proliferation with
bacterial growth stimulation (Spaepen et al. 2009).
11.5.4.2
Cytokinins
Cytokinins consist of a group of molecules, of which zeatin is the major representative (Fig. 11.2). These compounds have the capacity to induce division of plant
cells in the presence of auxin. Plants use ADP and ATP as substrates for cytokinin
biosynthesis while bacteria use AMP. In all cases, an isopentenyl side chain is
incorporated at the N6 position of the adenine ring. The balance between the
amounts of auxin and cytokinin determines whether root or shoot differentiate
from callus tissue. High auxin promotes root differentiation whereas high cytokinin
promotes shoot morphogenesis. Equimolar concentrations induce cell proliferation.
Cytokinins are synthesized in root tips and developing seeds. They are transported
to the shoot where they regulate important processes such as chloroplast development, leaf expansion, and delay of senescence. Exogenous application of cytokinins
to the plant can cause profuse lateral branching.
Many rhizosphere bacteria can produce cytokinins, e.g. Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Erwinia, Pantoea agglomerans, Pseudomonas,
Rhodospirillum rubrum, Serratia, and Xanthomonas (Garcı́a de Salome et al.
2001). The spectrum of cytokinins produced by rhizobacteria is similar to that
produced by the plant (Barea et al. 1976; Garcı́a de Salome et al. 2001; Frankenberger
and Arshad 1995), of which isopentenyladenine, trans-zeatin, cis-zeatin, and their
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ribosides are the most commonly found. Concerning the mechanism of action, one
speculates that cytokinin produced by rhizosphere bacteria becomes part of the
plant cytokinin pool, and thus influences plant growth and development.
Evidence for a role of cytokinin of rhizosphere bacteria in plant growth promotion has been published. Garcı́a de Salome et al. (2001) have produced mutants of
P. fluorescens strain G20–18 which produce reduced amounts of cytokinin and
normal amounts of auxin. In contrast to the wild-type strain, the mutants are unable
to promote growth of wheat and radish plants (Garcı́a de Salome 2000). Garcı́a de
Salome and Nelson (2000) showed that cytokinin production is linked to callus
growth of tobacco and suggested that this test can be used as a screening method for
cytokinin-producing bacteria.
For the pathogen Agrobacterium tumefaciens, the ability to produce auxins and
cytokinins is a virulence factor. The strain produces crown galls. The genes for
production of auxins and cytokinins are transferred to the plant and incorporated in
its DNA (Spaink et al. 1998). Another bacterium from this genus, A. rhizogenes,
modifies cytokinin metabolism causing the appearance of masses of roots instead of
callus from infection site (Hamill 1993).
11.5.4.3
Gibberellins (GAs)
These hormones consist of a group of terpenoids with 20 carbon atoms, although
active GAs only have 19 carbon atoms (Fig. 11.2). This group of compounds
consists of over 120 different molecules. GAs are mainly involved in cell division
and cell elongation within the subapical meristem, thereby playing a key role in
internode elongation. Other processes affected by these hormones are seed germination, pollen tube growth, and flowering in rosette plants. Like auxins and cytokinins, GAs mainly act in combination with other hormones.
Hardly anything is known about gibberellin synthesis in rhizosphere bacteria.
Bacteria that produce gibberellins, such as A. brasilense, A. lipoferum, Bacillus
species, Bradyrhizobium japonicum, and Rhizobium phaseoli, secrete them in
the rhizosphere (Frankenberger and Arshad 1995; Gutiérrez Manero et al. 2001;
Rademacher 1994).
The mechanism of plant growth stimulation by gibberellins is still obscure.
Fulchieri et al. (1993) speculate that gibberellins increase root hair density in root
zones involved in nutrient and water uptake.
11.5.5 Stress Controllers
Plants can be subject to stress. Bacteria which can decrease plant stress are treated
here.
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Abscisic Acid (ABA)
ABA is a 15-carbon compound (Fig. 11.2) and, like ethylene, it is involved in plant
responses to biotic and abiotic stresses. It inhibits seed germination and flowering.
It is involved in protection against drought, salt stress, and toxic metals. It also
induces stomatal closure.
ABA can be produced by several bacteria such as A. brasilense (Cohen et al.
2008) and B. japonicum (Boiero et al. 2007). The effect of inoculation with ABAproducing bacteria on plant growth is experimentally poorly underpinned. Since
ABA inhibits the synthesis of cytokinins (Miernyk 1979) it was speculated that ABA
increases plant growth by interfering with the cytokinin pool (Spaepen et al. 2009). It
could also alleviate plant stress by increasing the root/shoot ratio (Boiero et al. 2007).
11.5.5.2
Ethylene (ET)
Ethylene, the gaseous hormone (Fig. 11.2), is best known for its ability to induce fruit
ripening and flower senescence. It generally inhibits stem elongation in most dicots
favoring lateral cell expansion and leading to swelling of hypocotyls. However, it
promotes growth in submerged aquatic species. ET also breaks seed and bud dormancy. ET is synthesized under biotic stress conditions following infection by pathogens, as well as by abiotic stress conditions such as drought. It is therefore also known
as the stress hormone. In the plant, ethylene is produced from S-adenosylmethionine
(SAM) which is enzymatically converted to 1-aminocyclopropane-1-carboxylate
(ACC) and 50 -deoxy-50 methylthioadenosine (MTA) by ACC synthase.
The enzyme ACC deaminase is present in many rhizosphere bacteria. Such
bacteria can take up ACC secreted by the plant root and convert it into a-ketobutyrate
and ammonia. This results in the decrease of ACC levels, and therefore also of
ethylene levels in the plant and in decreased plant stress.
Inoculation of plants with ACC deaminase producing bacteria can protect plants
against stress caused by flooding, salination, drought, heavy metals, toxic organic
compounds, and pathogens (Glick 2005; Glick et al. 2007a; Glick et al. 2007b;
Belimov et al. 2005).
In addition to a direct role of ethylene on plant growth, this hormone can also act
as a virulence factor and a signaling molecule in plant protection against pathogen
attack. Ethylene production was reported to act as a virulence factor for bacterial
pathogens, e.g., P. syringae (Weingart and Volksch 1997; Weingart et al. 2001).
Furthermore, ethylene acts as a signaling compound in induced systemic resistance
(ISR) caused by some rhizobacteria (Van Loon 2007).
11.6
Bacteria Which Can Control Plant Diseases
Many plant diseases are caused by fungi. Some beneficial bacteria can reduce such
diseases.
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11.6.1 Introduction to Biocontrol Microbes
The story of microbes which can protect plants against pathogens begins with the
discovery by Schroth and Hancock (1982) of so-called suppressive soils. In these
soils, pathogens can be present without causing diseases to the normally susceptible
plants. These soils contain microbes which protect the plant from the pathogen.
When the disease-suppressing microbe is absent we speak about “conducive soils”
in which pathogens can cause diseases. A small amount of suppressive soil can,
when mixed with conducive soil, make the latter suppressive.
Many of the disease-suppressive microbes that have been isolated belong to the
genera Pseudomonas and Bacillus. Fluorescent pseudomonads can easily be
detected on Fe3þ poor medium because, under Fe3þ limiting conditions, e.g., in
Kings medium B (King et al. 1954), they secrete a siderophore, i.e., a molecule
which sequesters Fe3þ. In the case of fluorescent pseudomonads, (one of) the
siderophore(s) is pyoverdin (Fig. 11.2).
11.6.2 Mechanisms of Biocontrol
Researchers have focused on characterizing the mechanisms involved in biocontrol
operating under different experimental situations. In all cases, pathogens are antagonized by the presence and activities of other organisms they encounter (Haas and
Défago 2005; Pal and Gardener 2006; Alabouvette et al. 2006; De Weert and
Bloemberg 2007). The most important pathogens are fungi, but there are also
some pathogenic bacteria and nematodes. The modes of action of beneficial microorganisms can be based on either a direct or an indirect antagonism. However, both
mechanisms are not mutually exclusive as they have been frequently described as
co-occurring within the activity of the same BCA (Castoria and Wright 2009).
Direct antagonism results from physical contact and/or from a high degree of
selectivity of the mechanism(s) expressed by the BCA(s), in relation to the pathogen, i.e., parasitism and predation, production of antibiotics, and signal interference. In contrast, indirect antagonism results from activities that do not involve
sensing or targeting of a pathogen by the BCA(s), i.e., competition for nutrients and
niches, production of siderophores, and ISR.
11.6.2.1
Antibiosis
Antibiotics produced by microorganisms have been shown to be particularly effective in suppressing plant pathogens and diseases. Most biocontrol strains of Pseudomonas spp. with a proven effect in plant bioassays produce one or several
antibiotic compounds, e.g., P. fluorescens strains CHAO (Laville et al. 1998;
Haas et al. 2000) and Pf-5 (Thompson et al. 1999) produce complex cocktails of
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these secondary metabolites. In vitro, these antibiotics inhibit the growth of the
fungal pathogen, and for this reason, strains acting through antibiosis are usually
identified by screening them for antagonistic activity on plates on which the target
pathogen is also inoculated (Lugtenberg and Bloemberg 2004).
The contribution of antibiotics to biological control of root disease is documented in five experimental steps (1) Purification and chemical identification of the
antibiotic compound. (2) Detection and quantification in the rhizosphere of the
secondary metabolite. (3) Identification and characterization of the structural and
principal regulatory genes controlling the expression of the antibiotic compound.
(4) Poor biocontrol strains can acquire biocontrol activity by the introduction of
antibiotic biosynthetic genes not present in the original strain. (5) Detection of the
expression of the antibiotic biosynthetic genes through the use of detectable
reporter genes fused to structural genes for antibiotic biosynthesis (Haas and Keel
2003).Well-characterized antibiotics (Fig. 11.2) with biocontrol properties include
phenazines (Phz), phloroglucinols (Phl), pyoluteorin, pyrrolnitrin, hydrogen cyanide (HCN), cyclic lipopeptides (Perneel et al. 2008; Keel et al. 1992; Thomashow
and Weller 1988; Haas and Keel 2003; Raaijmakers et al. 2006), and the most
recently discovered 2-hydroxymethyl-chroman-4-one (Kang et al. 2004), D-gluconic
acid (Kaur et al. 2006), and 2-hexyl-5-propyl resorcinol (HPR) (Cazorla et al.
2006).
Phenazines are analogs to flavin coenzymes, inhibiting electron transport, and
are known to have various pharmacological effects on animal cells (Ran et al.
2003). 2-4-Diacetylphloroglucinol, the best known Phl compound, causes membrane damage to Pythium spp. and is particularly inhibitory to zoospores of this
oomycete (De Souza et al. 2003). Gleeson et al. (2010) provided evidence that Phl
acts through impairing the function of mitochondriae.
Dikin et al. (2007) reported that pyrrolnitrin causes the loss of mitochondrial
activity in the fungal cytoplasm, inhibiting succinate oxidase and NADH-cytochrome
reductase. Pyrrolnitrin also interferes with cellular processes such as oxidative
stress, blockage of electron transport as well as inhibition of DNA and RNA
synthesis (Dikin et al. 2007).
The cyanide ion derived from HCN is a potent inhibitor of many metalloenzymes, especially copper-containing cytochrome c oxidases (Blumer and Haas
2000). Finally, cyclic lipopeptides have surfactant properties and are able to insert
themselves into membranes and perturb their function, resulting in broad antibacterial and antifungal activities (Haas and Défago 2005; Perneel et al. 2008).
Recently, Mazzola et al. (2009) have shown that in the wheat rhizosphere the cyclic
lipopeptides viscosin and massetolide not only protect the plant against fungi but
also against protozoan predation. In fact, the protozoa Naegleria americana derepresses the syntheses of these antibiotics. To our knowledge, no mode of action
has been described for pyoluteorin, HPR, and D-gluconic acid.
The syntheses of antifungal metabolites (AFMs) are extremely sensitive to
environmental conditions in the rhizosphere, e.g., soil mineral content, oxygen
tension, osmotic conditions, carbon sources, as well as fungal, bacteria, and plant
metabolites can all influence the expression of secondary metabolites (Haas and
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Keel 2003; Lugtenberg and Bloemberg 2004; Duffy and Défago 1999; Van Rij
et al. 2005).
11.6.2.2
Predation and Parasitism
Soilborne bacteria and fungi are able to produce extracellular enzymes such as
chitinases, ß-1-3 glucanases, lipases, cellulases, and proteases. These lytic enzymes
can hydrolyze a wide variety of polymeric compounds, including chitin, proteins,
cellulose, hemicelluloses, interfering with pathogen growth and/or activities. Production and secretion of these enzymes by different microbes can result in biocontrol abilities (Markowich and Kononova 2003). Ordentlich et al. (1998) showed that
chitinase of Serratia marcescens is involved in the biocontrol of Sclerotium rolfssi.
Beta-1,3-glucanase contributes significantly to the biocontrol activities of Lysobacter enzymogenes strain C3 against Bipolaris leaf spot caused by Phytium spp.
(Palumbo et al. 2005). Chitinases of the mycoparasitic Trichoderma species have
also been shown to play an important role in biocontrol and antagonistic activity
against phytopathogens (Harman et al. 2004), including R. necatrix (Hoopen and
Krauss 2006). Sometimes, these enzymes act synergistically with antibiotics playing an important role in the antagonistic effect on phytopathogenic fungi
(Schirmb€
ock et al. 1994; Fogliano et al. 2002). Predation and parasitism are not
always related to biocontrol, as it is the case for the fungus eater C. fungivorans, of
which the major mechanism of action for controlling tomato foot and root rot
(TFRR) probably is competition for space and nutrients and niches (Kamilova
et al. 2007).
11.6.2.3
Competition for Nutrients and Niches
To successfully colonize the rhizosphere, a microbe must effectively compete for
the available nutrients. Competition for nutrients and niches (CNN) between
pathogens and beneficials has been shown to be important for limiting disease
incidence and severity (Kamilova et al. 2005). Enrichment for enhanced competitive tomato root tip colonizers was used to select for bacteria, not producing
antibiotics, which control TFRR by CNN (Fig. 11.1) (Kamilova et al. 2005). The
enhanced competitive root tip colonizers grow efficiently on root exudates. However, Kamilova et al. (2005) observed that these two characteristics were not
sufficient for biocontrol, as one of the best competitive root-tip-colonizing strains
did not control TFRR.
A similar enrichment procedure was used by Pliego et al. (2007) for the isolation
of competitive avocado root tip colonizers strains displaying biocontrol ability
against R. necatrix. Two Pseudomonas sp. strains, P. pseudoalcaligenes AVO110
and P. alcaligenes AVO73, were selected for their efficient colonization abilities.
However, only AVO110 demonstrated significant protection against avocado white
root rot. Further analysis revealed that both strains colonize different sites on the
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C. Pliego et al.
root: biocontrol strain AVO110 was observed to colonize the root at preferential
penetration sites for R. necatrix infection (intercellular crevices between neighboring plant root epidermial cells and root wounds) while P. alcaligenes AVO73 was
predominantly found forming dispersed microcolonies over the root surface and in
the proximity of lateral roots, areas not colonized by this pathogen (Pliego et al.
2008). These results strongly suggest that biocontrol bacteria acting through CNN
must efficiently colonize the same mini-niche as the pathogen.
Competition for ferric iron ions is a well-documented example of competition of
biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986, Lugtenberg
and Bloemberg 2004). This topic has been treated under 5.2.
11.6.2.4
Induced Systemic Resistance
Some PGPR have been identified as potential ISR elicitors, for their ability to induce
resistance in plants toward pathogenic fungi, bacteria, and viruses (Van Loon et al.
1998; Van Loon 2007). The inducing rhizobacteria triggered a reaction in the plant
roots that gave rise to a signal that spread systemically throughout the plant and
enhanced the defensive capacity of distant tissues to subsequent infection by the
pathogen (Van Loon 2000). ISR is distinct from systemic acquired resistance (SAR)
in several key physiological and biochemical phenotypes that are best defined in
A. thaliana (Van Wees et al. 1997). Studies with A. thaliana mutants indicated that
the jasmonate/ethylene-inducible defense pathway is important for ISR.
Many bacterial determinants induce ISR, i.e., flagella, siderophores (pycholin
and pyocyanin), LPS, salicylic acid (Van Loon 2007), cyclic lipopeptides (Ongena
et al. 2007), N-acyl homoserine lactone (AHL) molecules (Shuhegger et al. 2006),
the bacterial volatile 2,3-butanediol produced by Bacillus spp. (Ryu et al. 2003),
and antibiotics such as Phl (Lavicoli et al. 2003). In several ISR-competent strains
of fluorescent pseudomonads, it has been difficult to identify specific ISR elicitors,
possibly because a combination of siderophores, O-antigen, and flagella might
account for the ISR effect (Bakker et al. 2003). It has also been shown that several
Pseudomonas spp. are able to induce ISR in a wide range of plants toward different
pathogens (Van Loon 2007). Generalization of the signal transduction pathways
that are involved in ISR are further complicated by the fact that an ISR response to a
given PGPR depends on the plant species and cultivar. For example, in Arabidopsis
thaliana, the PGPR strain P. fluorescens WCS417r elicited ISR on all ecotypes
examined, except ecotypes Wassilewskija and RLD (Van Wees et al. 1997).
11.6.2.5
Signal Interference
In Gram-negative bacteria, one type of communication system functions via
small, diffusible N-acyl homoserine lactone (AHL) signal molecules (Fig. 11.2).
Such a regulatory system allows bacteria to sense the density of cells of their own
kind and to express target genes in relation to their cell density. This cell–cell
11
Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
319
communication mechanism regulates a variety of physiological processes, including
swarming, swimming and twitching motilities, production of pathogenicity/virulence factors, and rhizosphere colonization (Gray and Garey 2001; Miller and
Bassler 2001). Several groups of AHL-degradation enzymes have recently been
identified in a range of organisms, including bacteria and eukaryotes. Expression of
these enzymes was identified to interfere with the quorum-sensing system of pathogenic bacteria. E. carotovora produces and responds to AHL quorum-sensing
signals to regulate antibiotic production and expression of virulence genes, whereas
B. thuringiensis strains abolished the accumulation of the AHL signal by the
expression of a AHL-lactonase, which is a potent AHL-degrading enzyme. In plants,
B. thuringiensis significantly decreased the incidence of E. carotovora infection and
symptoms development of potato soft rot caused by the pathogen (Dong et al. 2004).
Discovery of these enzymes has not only provided a promising means to control
bacterial infections, but also represents new challenges to investigate their roles in
host organisms and their potential impacts on ecosystems (Dong et al. 2004).
11.6.3 Role of Root Colonization in Biocontrol Mechanisms
Selection of bacterial biocontrol strains by their ability to inhibit fungal growth
in vitro is not always correlated with efficient control of plant diseases. Different
steps are known to be involved in root colonization (1) attachment to the plant
surface, in which flagella and different types of pili can be involved (De Weert et al.
2004b) and (2) surface motility which confers potential benefits to bacteria in the
rhizosphere, including increased efficiency in nutrient acquisition, avoidance of
toxic substances, ability to translocation to preferred hosts, and ability to access
optimal colonization niches (Andersen et al. 2003). Motility has been reported to
depend on the soil type, the plant, and the bacterial strains used (Weller and
Thomashow 1994). Pliego et al. (2008) reported that strains differing in surface
motility can show differences in the architecture of the biofilms developed by these
strains in the rhizosphere which, at the same time, can reflect differences in their
root colonization patterns. As we explained earlier, in case of BCAs acting through
CNN, colonization strategies of the biocontrol strain must be correlated with
penetration sites used for the phytopathogenic fungi to infect the root.
In the case of AFM production, efficient root colonization is essential for the
delivery of the AFM along the root system at the right time and place (Lugtenberg
et al. 2001; Haas and Keel 2003). Insertional mutagenesis studies demonstrated that
colonization can be a limiting step in some biocontrol strains. P. chlororaphis
PCL1391 controls TFRR through the production of the antifungal metabolite
phenazine-1-carboxamide (PCN) (Fig. 11.2). Nonmotile mutants of this strain
impaired in root colonization, but not in production of extracellular metabolites,
did not show biocontrol activity (Chin-A-Woeng et al. 2000).
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C. Pliego et al.
In the case of microorganisms inducing the ISR response on the root, a decrease
in root colonization does not clearly result in loss of biological control, i.e.,
P. fluorescens WCS365, which acts through ISR (Kamilova et al. 2005), colonization of seedlings for a brief period is sufficient to induce ISR in the whole plant
(Dekkers et al. 2000). As is mentioned earlier, certain antifungal metabolites can
induce ISR. This can explain the biocontrol activity of poorly colonizing PGPRs
which produce antibiotics (Gilbert et al. 1994).
11.6.4 Mycelium Colonization by Bacteria
As mentioned previously, biocontrol bacteria in the rhizosphere compete for nutrients and niches with endogenous microorganisms. Therefore, to gain a better
understanding of how biological control functions in the rhizosphere, the interaction with other microbes, such as root pathogenic fungi, must be taken into account.
It has become clear that biocontrol bacteria can interact with phytopathogenic fungi
in a variety of ways, all converging on the same objective, i.e., to derive nutrition
from these fungi (Leveau and Preston 2008).
This interaction can be split into several stages including (1) detection of the
fungal host, (2) attachment to the fungal cells, and (3) growth of bacteria on biotic
fungal surfaces (Hogan et al. 2009). Microscopic visualization of bacterial–fungal
interactions showed that at least some antagonistic bacteria exhibit chemotaxis
toward fungal exudates allowing bacteria to congregate around populations of
fungi in soils. For example, fusaric acid (Fig. 11.2), a secondary metabolite secreted
by Fusarium hyphae, acts as a chemoattractant for cells of biocontrol strain
P. fluorescens WCS365, which can actively move toward and colonize the surface
of fungal hyphae (De Weert et al. 2004a). Bacteria showing chemotaxis to specific
fungal compounds may use a variety of mechanisms to reach and attach to the fungus,
including pili, exopolysaccharides, and biosurfactans. The biosurfactants reduce
surface tension, enhance swarming motility, and facilitate contact between bacteria
and fungal cells (Nielsen and Sorensen 1999; Braun et al. 2001). Once bacteria reach
the fungus, they may scavenge nutrients from the fungal cell wall, consume products
secreted by the fungus, or induce lysis of the fungal cells, thereby liberating the
intracellular contents for consumption by the local bacterial population.
Bacterial communities associated with fungi seem to be under selection to
develop fungi-specific traits that confer a competitive advantage during colonization of fungal surfaces. Such traits could be the ability to detect fungus specific
compounds, the utilization of nutrients that are specific to, or particularly abundant
in, fungal exudates, or the ability to tolerate or suppress the production of antibacterial metabolites by fungal cells.
Several studies support the idea that hyphal colonization by bacteria can play an
important role in biocontrol activity (Bolwerk et al. 2003, Pliego et al. 2008). It has
been shown that the ability of P. fluorescens WCS365 to inhibit pathogenic activity,
survival, and germination of the pathogen significantly contributes to the reduction
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
321
of TFRR after introduction of this bacterium into the plant nutrient solution
(Kamilova et al. 2008). Co-inoculation of the biocontrol strain P. pseudoalcaligenes AVO110 with R. necatrix (the causal agent of avocado white root rot)
showed that AVO110 utilizes hyphal exudates more efficiently for proliferation
than the nonbiocontrol strain P. alcaligenes AVO73 (Pliego et al. 2008). Although
it is not always clear whether bacterial consumption of fungal exudates contributes
to reduction of the disease (Kamilova et al. 2007), bacteria can inhibit the growth of
harmful fungi by feeding on them, thus supporting their own growth, which in turn
leads to a better biocontrol activity (Leveau and Preston 2008).
While several studies have been focused on identifying bacterial genes involved
in root colonization, little attention has been paid to genes involved in interactions
with fungi. Bacterial genes involved in the interaction established between biocontrol strain P. putida 06909 and the phytophatogenic oomycete Phytophthora have
been identified using IVET. Several P. putida promoters were induced during
the growth of this biocontrol bacterium on the surface of the fungus, corresponding
to genes involved in carbon catabolism, amino acid/nucleotide metabolism,
and membrane transport processes (Lee and Cooksey 2000; Ahn et al. 2007)
(Table 11.3). Separate studies showed that genes involved in trehalose utilization
may be important for bacterial growth when associated with fungi. P. fluorescens
sp. have been shown to induce genes involved in trehalose metabolisms when
exposed to fungal culture supernatant, and the presence of this sugar enhances
inhibition of Pythium debaryanum in a radial growth assay (Gaballa et al. 1997;
Rincón et al. 2005).
Recently, Pliego (2008) used STM, a powerful genomic mutagenesis technique
which has been widely applied to bacterial pathogens (Hensel et al. 1995; Shea
et al. 2000; Autret and Charbit 2005) to identify genes involved in growth and
survival of a biocontrol Pseudomonas strain in fungal exudates. These genes were
predicted to be involved in central metabolism, regulation of the second messenger
cyclic (c)-di-GMP, and bacterial protection against fungal defenses.
Table 11.3 Pseudomonas proteins upregulated during colonization of Phythophthora myceliaa
Energy metabolism
Nucleotide biosynthesis
Membrane proteins/
transporters
Nitrogen regulatory protein PII-2 Carbamoyl-phosphate synthase C4-dicarboxylate transport
protein
Flavin-dependent oxidoreductase Phosphoribosylaminoimidazol Arginine-/ornithine-binding
(AIR) synthetase
protein
Malic enzyme
Carbamoylphosphate
Leucine-,isoleucine-,
synthetase large subunit
valine-binding protein
Succinate-semialdehyde
ABC transporter
dehydrogenase
Probable glyceraldehydeOuter membrane porins
3-phosphate dehydrogenase
Ribulose-phosphate-3-epimerase
Transaldolase
a
Adapted from Lee and Cooksey (2000) and Ahn et al. (2007)
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C. Pliego et al.
In summary, further studies must be performed to identify bacterial genes
involved in fungal colonization or acquisition of nutrients from fungi. This will
gain a broader view on genes and mechanisms involved in biological control and in
future design of new and improved BCAs.
One of the most exciting aspects is that genes similar to those involved in
colonization of fungal hyphae were also identified in other IVET or STM assays
involving phylogenetically diverse pathogenic and nonpathogenic microorganisms
residing in diverse complex environments, in particular plant or animal host. These
results suggest that similar or common mechanisms might be involved in processes
as different as controlling and causing diseases. The identification of bacterial genes
necessary for biocontrol of fungal pathogens will expand our ability to design new
biocontrol strategies and, above all, feed our growing appreciation of the enormous
diversity and adaptability of bacteria.
11.6.5 Resistance of the Pathogen Toward Biocontrol Agents
Although there are hardly any reports about pathogenic fungi that have become
resistant against BCAs, it is good to consider this possibility. It is clear that the
mechanism of action used by the BCA determines the possible resistance strategies.
Since little is known about the interaction between the pathogenic fungus and the
plant when ISR is used as the biocontrol mechanism, it is difficult to predict
possible defense strategies for this case. When CNN is used, it is hard to envisage
resistance strategies of the pathogen.
In contrast, for antibiosis as a mechanism of biocontrol several defense strategies
can be envisaged. To do this, it is best to look at the much better studied resistance
mechanisms that bacteria are using against antibiotics. Here, known resistance
strategies can be based on (1) detoxification of the antibiotic, e.g., by degradation
or modification, (2) modification of the target of the antibiotic, (3) making penetration to the target molecule more difficult, e.g., by making entry pores narrower or by
shielding the surface with an additional less penetrable layer, and (4) actively
pumping the antibiotic out of the cell.
Duffy et al. (2003) published an excellent review about defense mechanisms
used by pathogens against microbial antagonism. Detoxification of the antifungal
compound 2,4-diacetylphloroglucinol produced by Pseudomonas species has been
reported. Schouten et al. (2004) reported the screening of 76 plant-pathogenic and
41 saprophytic F. oxysporum strains for sensitivity to 2,4-diacetylphloriglucinol.
They found that 18 and 25%, respectively, of these strains were tolerant to the
antibiotic. Of course, an antibiotic-producing strain should protect itself against
the antibiotic it produces. Indeed, Bacillus subtilis strain UW85 which produces the
antibiotic zwittermycin A can inactivate the antibiotic by acetylation (Milner et al.
1996).
Another mechanism that can be used by a pathogenic fungus is efflux of the
antibiotic. Upon exposure to phenazines, Botrytis cinerea induces an efflux pump
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
323
for the antibiotic. Mutants lacking pump activity are more sensitive toward the
antibiotic (Schoonbeek et al. 2002).
A very interesting mechanism of defense is the ability of a pathogen to repress
the biosynthesis of the antibiotic produced by the BCA. Duffy and Défago (1997)
found that fusaric acid (Fig. 11.2), a small molecule secreted by many Forl strains,
represses the synthesis of Phl in the biocontrol bacterium P. fluorescens CHAO by
repression of the phlA promoter, a process for which an intact phlF gene is needed.
Van Rij et al. (2004) observed that fusaric acid also suppresses the synthesis of
another antibiotic, namely, PCN produced by P. chlororaphis strain PCL1391. This
inhibition correlates with reduction of the level of the AHL autoinducer N-hexanoylL-homoserine lactone. The inhibition in P. chlororaphis strain PCL1391 takes place
at or before the level of AHL production. It is striking to see that in two different
Pseudomonas biocontrol strains inhibition of antibiotic production by fusaric acid
has evolved, although at different molecular levels. Apparently, these mechanisms
have evolved independently because of the need to protect against antibioticproducing bacteria. It should be noted that P. fluorescens strain CHAO does not
produce AHL and therefore the mechanism to suppress Phl production at the level
at or before AHL production could not be used in this strain.
In practice, there are very few examples of resistance of pathogens against BCAs
(Duffy et al. 2003). This may have several reasons, for example, that a BCA uses a
mechanism to which no resistance exists or that it uses several different mechanisms to which no cross-resistance exists. The relevance of resistance in biocontrol
was studied by Mazzola et al. (1995). They screened a collection of 66 isolates of
the pathogen Gaeumannomyces graminis var. tritici against the antibiotics Phl and
PCA, which are produced by many biocontrol strains which act through antibiosis.
A large variation in sensitivity was found. Most importantly, it appeared that the
Phl- or PCA-producing Pseudomonas strains could not control take-all caused by
the Phl- or PCA-resistant G. graminis var. tritici isolates, respectively.
11.7
From Laboratory to Industrial Application
It is important to realize that a beneficial bacterium, which is active under laboratory conditions, not necessarily is a good basis for a commercial product. Here, we
discuss criteria that industrial products should fulfill.
11.7.1 Introduction
The application of beneficial microbes for plant growth stimulation and biocontrol
makes horticulture and agriculture sustainable and will, in contrast to many chemicals, not be a burden for the ecology and for the next generations of human beings.
This notion, supported by public, politicians, and some supermarkets, is a major
324
C. Pliego et al.
driving force for the agricultural and horticultural industry to use, where possible,
environmentally friendly products for plant growth stimulation and crop protection.
In this section we will discuss how, starting from promising beneficial microbes
with known mechanisms of action, the industry will make the decision to carry out
further research and which steps are required to develop a marketable product.
Because this chapter focuses mainly on biocontrol microbes, we discuss only
biocontrol products in this section. Furthermore, rules about registration as a
product differ among countries. We will mainly focus on guidelines of the European
Union (EU).
11.7.2 Evaluation of Opportunities
How does the biocontrol industry come to the decision which strain should be used
to make it a profitable product? What are the prerequisites for such a decision?
What does the grower need? How does the development of a plant protection
product occur? How can such a product be protected, registered, and commercialized? The biocontrol industry keeps constant contact with growers, the current
users of their products. Collecting data on existing and emerging diseases of crops
and search for solutions against these diseases is the starting point for decision
making. The size of the market is another important factor. Therefore, the market
potential for a new microbial product will be evaluated in terms of market size in
relation to (1) the importance of the crop(s) and (2) the severity and geographical
spread of the disease. What are the present biological and/or chemical products for
disease prevention? Who are the competitors? How strong are they? Would the new
product have a chance to successfully penetrate the market?
11.7.3 Industry Takes over
In case the above-mentioned evaluation is positive, a good strain has to be found. If
the biocontrol industry has no useful strains available, it can approach academic
parties to evaluate whether they have promising strains that are effective against the
target disease/pathogen combination. Academic research, as described in the previous paragraphs, is usually directed toward the discovery of the potential plantbeneficial strains by using approaches such as screening for antagonistic properties,
studying root colonization, and identifying traits and metabolites involved in
biocontrol and plant growth promotion. Most of this research is done in vitro or
in planta under laboratory, growth chamber, or experimental greenhouse conditions. Academic scientists usually focus on one pathogen/crop model system as
such experiments can easily be made suitable for publication. Industry should
ensure that the selected strain is safe for environmental release and should come
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
325
to an agreement with the owner on intellectual property rights and the degree of
exclusivity and/or licensing.
11.7.4 Industrial Research
In contrast to the usually limited plant experiments done by academic researchers,
industry would like to develop a product applicable on a broader range of crops and/
or pathogens. This implies further research as well as the development of a suitable
formulation for the strain. After positive evaluation of marketability of the potential
biocontrol product, the following considerations are taken into account: strain
safety and efficacy, potential for upscale production and product development,
and registration costs.
11.7.5 Strain Safety
The beneficial strains have usually already been taxonomically identified in an
early state of the research. According to guidelines (Anonymous 1998) the strain
can then be classified in risk groups which predict their degree of safety. Before a
BCA can be registered as a product, cost-intensive, time-consuming pathogenicity
tests have to be carried out. There is a good reason to do this since several strains
isolated as potential BCAs are potential human pathogens (Berg et al. 2005;
Egamberdieva et al. 2008). Thus, strain identification is crucial for safe applications in agriculture and horticulture. According to European Commission Directive
2001/36/EC amending Council Directive 1991/414/EEC, concerning the introduction of plant protection products on the market, the best available technology
should be used to identify and characterize the microorganism at the strain level.
The use of molecular biological techniques, mostly at the DNA level, allows the
identification of potential pathogens which subsequently will be excluded from
further research. It should be noted that a promising new tool for future evaluation
of the pathogenic potential of putative biocontrol strains could be the use of an
assay with the nematode Caenorhabditis elegans (Zachow et al. 2009). Work will
only be continued with strains from the risk group 1 (Anonymous 1998). In this
stage also evaluation of the mode of action of the beneficial strains is very
important, because it will give a clue to whether this would cause additional
studies and, consequently extra costs, for registration of the biocontrol product.
For example, for strains with mechanisms of actions based on production of new
antibiotics and/or other toxic compounds, the regulatory authority will ask to
provide very detailed investigations on metabolite production, toxicological and
residue studies.
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C. Pliego et al.
11.7.6 Large-Scale Production and Formulation
Often the high cost of production for most of BCAs is a limiting factor for
commercialization of a biocontrol product (Fravel 1998). High costs of the growth
substrate, low productivity of the microbe, or limited economies of scale could be
the reasons to stop development (Fravel et al., 2005). One of the goals of an
industrial production unit is to minimize the fermentation costs and to produce
the highest quantities of the microbe without negatively influencing the important
traits involved in biocontrol such as (1) proliferation of spores in case of sporeforming bacteria and fungi, (2) ability to colonize rhizosphere or phyllosphere, or
(3) competition with phytopathogenic organisms. Multiple tests on cell viability
and plant root colonization should be performed in order to optimize a manufacturing process and to produce a good quality product.
Beneficial microbes are formulated in order to preserve the BCA, to deliver the
microbes to their targets and, after their delivery, improve their activity (Burges
1998). Formulation usually consists of the addition of a combination of various
additives, such as carriers, preservatives, nutrients, etc. Furthermore, the choice of
dry or liquid formulation to be used depends on the biological and physical properties of the microorganism, and on its abilities to survive the formulation process and
to maintain the desired properties for a certain period of time (Burges 1998).
Evidently, for producing powder and granular formulations, spore-forming
strains such as Bacillus have an advantage over non-spore-forming strains such as
Pseudomonas which have to be formulated as vegetative cells. Spores are more
robust and resistant to elevated temperature and high concentrations of chemicals
that are part of the spray-drying process. Moreover, the shelf life of biological
products based on bacterial spores can be up to 1–3 years. A disadvantage of the use
of spores is that, after application, they need time to return to the metabolic active
stage of a vegetative cell.
Vegetative cells of nonspore formers are more sensitive than spores toward dry
formulation processes. This makes dry formulation of vegetative cells more expensive than formulation of spores. For example, it has been shown for Pseudomonas
by Validov et al. (2007) that freeze-drying provides a more stable formulation
product with a longer shelf life than the less expensive spray drying. A liquid
formulation can be an option for vegetative cells. However, it can be a challenge to
keep these bacterial cells in a low metabolic state in the presence of water for a
substantial amount of time without losing their viability.
For a seed company, seed coating with biocontrol strains is an interesting option.
Coating means here the introduction of BCAs during seed priming and/or seed
pelleting. In fact, this is a process for upgrading the value of seeds. An advantage
for the user is that the seeds do not have to be treated with the bioproduct
immediately prior to sowing by means of spraying or by soaking the seeds at site.
The choice of the formulation type is dependent not only on the biological
properties of the strains but also, in many cases, on the desired application methods
and existing irrigation systems. For example, for a drench application, slurry, spray
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
327
or soaking treatment of seeds, liquid or wettable powder products are more suitable.
A granular formulation would be appropriate for mixing with horticulture potting
mix or for distribution in-furrow (Fravel 1998).
11.7.7 Registration in the EU: Legal Basis and Requirements
Many biocontrol products have been registered. See Table 11.4 for examples.
Active microorganisms used in the plant protection products in the EU are regulated
according to the EU Council Directive 1991/414/EEC. This Directive provides
definitions of the plant protection products and requirements for their authorization
Table 11.4 Examples of active microorganisms registered as biopesticides in the EU and/or the
USA (as at March 1, 2010)
Active microorganism
Formulation Pathogen
Crop
type
Liquid,
Fusarium, Rhizoctonia,
Potato, vegetables,
Bacillus subtilis FZB24b
powder,
Pythium
ornamentals, turf
granule
Powder
Botrytis, Sclerotinia,
Vegetables,
Bacillus subtilis
Sphaerotheca macularis
soft fruits
QST 713b
Powder
Aspergillus, Pythium,
Cotton, vegetables,
Bacillus subtilis GB03b
Rhizoctonia, Fusarium
soybean
Liquid
Fusarium, Septoria,
Cereals
Pseudomonas
Tilletia
chlororaphis MA 342b
Powder
Fusarium
Vegetables
Streptomyces K61a,b
Powder
Rhizoctonia, Pythium,
Gliocladium catenulatum
Vegetables,
Phytophthora, Fusarium,
strain J1446a,b
herbs,
Didymella, Botrytis,
ornamentals,
Verticillium, Alternaria,
trees,
Cladosporium,
shrubs,
Helminthosporium,
turf
Penicillium, Plicaria
Powder,
Fusarium, Pythium,
Vegetables,
Trichoderma harzianum
granule
Rhizoctonia
ornamentals, turf
strain T-22a,b
Powder
Fusarium, Pythium,
Vegetables, turf
Trichoderma asperellum
Rhizoctonia
strain ICC012a,b
Powder,
Fusarium, Pythium,
Vegetables, turf
Trichoderma atroviride
granule
Rhizoctonia
strain IMI206040a
Fusarium, Pythium,
Vegetables
Trichoderma gamsii strain Powder
Rhizoctonia,
ICC080a,b
Verticillium
Trichoderma polysporum
Powder
Botrytis, Chondrostereum
Soft fruits,
strain IMI206039a
ornamentals
Liquid
Alternaria, Botrytis,
Canola, ornamentals,
Pythium oligandruma,b
Fusarium, Sclerotinia
vegetables
Liquid
Ophiostoma nova-ulmi
Elm
Verticillium albo-atrum
strain WCS850a,b
a
EU Annex I listed strains (http://ec.europa.eu/sanco_pesticides/public)
b
US-EPA registration (http://www.epa.gov/oppbppd1/biopesticides/ingredients)
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C. Pliego et al.
within the EU. Directive 1991/414/EEC was amended by Commission Directive
2001/36/EC regarding to the data requirements for the Annex I inclusion of microorganisms as active substances and national authorization of products (Annex II and
III, respectively, of the directive). Council Directive 2005/25/EC lays down the
Uniform Principles for evaluation and authorization of plant protection products
containing microorganisms.
Briefly, a registration dossier prepared in order to register a biopesticide in the
European Union must contain all requested information on the active microorganism and the product, such as the biological properties of the microbe, the physical
and chemical properties of the product, its safety for humans and for the environment, and its efficacy. In contrast to Europe, registration in the USA (http://www.
epa.gov) does not require the efficacy data. In Table 11.4, we provide examples of
microorganisms that have been registered as active substances in commercial
biopesticides registered in the EU and/or the USA.
Many physical and chemical studies of the product, the analysis of relevant
metabolites and potential toxins in the preparations, the analysis of residues on treated
plants and food, as well as toxicological and ecotoxicological studies should be
performed under Good Laboratory Practice (GLP) conditions. In most of the cases,
industry outsources these tasks to specialized GLP-certified laboratories and institutes. Toxicological studies include evaluation of acute toxicity studies performed on
certain laboratory animals such as rats, rabbits, and guinea pigs. Genotoxicity testing
includes both in vivo (carcinogenic) and in vitro (mutagenic) studies. Characteristics
such as impact of the product on the environment, particularly its effects on birds,
aquatic organisms, bees, and other nontarget arthropods, earthworms, and soil microorganisms, are analyzed by direct observation of the tested organisms under laboratory conditions. Information from the relevant literature on the ecology of biocontrol
microorganisms as well as closely related strains can help to evaluate the impact of
this strain on soil microflora by extrapolation. Providing this information therefore
can somewhat reduce laboratory studies on this subject. For example, there is a
number of useful publications about the analysis of microbial communities of various
soils after introduction of different bacterial strains with antagonism against Rhizoctonia solani (Adesina et al. 2009; Garbeva et al. 2004; G€otz et al. 2006; Scherwinski
et al. 2008), E. carotovora, and Verticillium dahliae (Lottmann et al. 2000). However,
this reduction of actual laboratory studies would be rather an exception, since the vast
majority of studies must be performed with the strain to be registered.
The efficacy part of the dossier is based on data obtained in the trials, performed
for registration purposes, as well as those carried out in the research laboratory. The
latter ones are considered only as preliminary data and treated by the experts
accordingly. In the EU, efficacy trials are required to be carried out by efficacy
testing organizations officially recognized by the national authorities. Efficacy tests
and analyses include field, glasshouse, or laboratory trials and tests to determine the
effectiveness and crop safety of plant protection products. Official Recognition is
also known as Good Experimental Practice (GEP). This depends on the national
regulations. Efficacy tests can be required to be performed during a number of
seasons and at different locations.
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In the framework of the European project on regulation of biological control
agents (REBECA, 2008), an analysis on registration issues of the most important
products (Annex I – listed) was performed. Data indicates that the mean registration
time for EU Annex I inclusion is 75 months. Country registrations vary widely and
range from a few months up to over 100 months, averaging around 24–36 months.
Overall registration cost for EU Annex I inclusion is estimated to be about
1,890,000 Euro; out of that 970,000 Euro are external costs (fees, etc.). The
breakdown of total costs to different kinds of tests averaged as efficacy tests
(21%), toxicological tests (43%), ecotoxicological studies (23%), and other
required studies (13%) (REBECA 2008). In case of use of several microbial strains
in one product (so-called microbial cocktails), registration costs will increase
because an individual dossier for each active microorganism must be submitted.
Registration strategy may include several options to minimize costs. For example, several companies that manufacture products with the same active microorganism(s) can unify into a task force team for inclusion of these organisms into
Annex I list and share costs of this procedure. In this case member companies also
share their studies and submit a joint dossier (Annex I) for the active microorganism. Individual access to certain protected data (mostly on acute toxicity and
analysis of metabolites) belonging to other companies can be an option to save
time for registration of some microorganisms, although financial considerations
must be taken into account.
11.7.8 Intellectual Property Rights and Patenting
Usually intellectual property rights on biocontrol strains belong to universities and
small companies in which the basic research was performed. If a biocontrol company decides to commercialize a strain, both parties should come to an agreement on
how the strain owner should be reimbursed. Protection is a rather sensitive subject
for the biocontrol industry. In Europe, patenting of biocontrol strains per se is not
possible due to the fact that these strains are naturally occurring living organisms.
This is why the subject of invention usually is a combination of the strain with the
bioproduct formulation(s) containing the viable cells or of the strain with applications against certain pathogenic organisms on certain crops. Another possibility for
patenting is a combination of the strain and its specific metabolites involved in
biocontrol activity and being produced during the fermentation and incorporated
into the bioproduct formulation. Patenting can take place in various stages of the
product development but should certainly take place prior to registration.
11.7.9 Marketing
Commercialization of the biocontrol products is not a simple task. First of all,
demonstration trials at sites should convince farmers that the proposed bioproduct
330
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can provide a sound protection which is preferentially better than, or at least
comparable with, other biological or chemical products (if these exist). Commercialization should also include a strong education element with emphasis on the
preventive character of the biopesticide. Also, the new product should be easy to
use. It would be ideal if the bioproduct can be integrated in grower’s routine
schemes for the use of pesticides. Last but not least, the price of the new biocontrol
product must be competitive and affordable for farmers without compromising the
desired sales margins.
11.7.10
Integrated Pest Management (IPM)
In 2005, biopesticides had a share of approximately US$ 300–600 million of the US
$ 35 billion agrochemical market (Cherry, 2005). Over the next 10 years, the market
for biopesticides is expected to grow to US$10 billion (GIA, 2008). This is mainly
due to the great public awareness of the risks of chemical fungicides and the clear
trend toward sustainable agriculture, both in the EU and the USA. A good example
of an effect of the public opinion is an activity of many supermarket chains and
retailers toward to pesticide poor or pesticide free food. Within GLOBALG.A.P.,
a private sector body, many food retailers and supermarket chains, farmers and crop
protection specialists collaborate in this respect. This organization sets standards
for the certification of agricultural products worldwide. The GLOBALG.A.P standard is primarily designed to reassure consumers about how food is produced on the
farm by minimizing detrimental environmental impacts of farming operations,
reducing the use of chemical inputs, and ensuring a responsible approach to worker
health and safety as well as animal welfare (www.globalgap.com).
Increasingly more attention is paid to IPM. This includes, among other means, a
balanced use of chemicals and biologicals in order to provide healthy food without
loosing or, preferentially, with even increasing productivity. Commercial biocontrol products must address not only efficacy and, broadly speaking, safety requirements, but also be compatible with existing agricultural practices, including the use
of fertilizers and chemical plant protection products. The ultimate intention of the
biocontrol industry is to minimize and eventually eliminate the use of chemicals by
replacing them with environmentally friendly biocontrol products.
11.8
Lessons from the Past to Create a Shining Future
In the past 3 decades, insight in fundamental aspects of microbe–plant interactions
has enormously increased, especially at the molecular level. Understanding of these
interactions at the molecular level is important for (1) improving the efficacy of
the applied microbes, (2) enhancing the registration process, and (3) marketing
microbes as reliable products to the users. In the recent past, mechanisms of biocontrol
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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation
331
through antibiosis have been elucidated, and the biocontrol mechanism CNN has
been proven to exist. The most obscure biocontrol mechanism known so far, ISR, is
presently being studied intensively and major breakthroughs may be expected in the
next decade. Plant growth promotion through hormones needs further attention.
In the past decade, endophytic microbes received a lot of interest, also for
applications as beneficial microbes. The possible advantage of the use of endophytes
is two-fold. (1) The plant’s interior may harbor a novel type of microbes. (2) Within
the plants, beneficial bacteria are much better protected against competition by
rhizosphere bacteria than when they are present in the rhizosphere. It should, however, be noted that a bacterium, which has been isolated as an endophyte, does not
automatically resume its endophytic lifestyle when applied to the plant’s exterior. In
addition, once inside the plant, it may have to compete with endogenous endophytes.
A bottleneck in the understanding of microbe–plant interactions is our limited
knowledge about the composition and function of root exudates. This requires a
thorough study in which experts in the fields of microbiology, plant physiology,
engineering, and soil science should work together. They should use the most
modern technology combined with knowledge of the facts from the literature. In
addition, a role of volatile organic compounds in biocontrol is becoming clear. The
characterization of these compounds and the elucidation of their functions should
become a new area for future research.
Many studied and commercialized biocontrol strains have been isolated only on
the basis of antibiotic activity whereas important traits such as root colonization and
compatibility with formulation and industrial practice were not taken into account.
Considering the complex and expensive process required to commercialize a plantbeneficial microbial product, it is crucial that, before large research efforts are
undertaken, one evaluates all steps of the process, from isolation strategy and
laboratory experiments to efficacy, registration, and marketing. Weak and expensive steps should be identified, evaluated and, if necessary, tested first. In this way a
lot of time can be saved if one wants to commercialize a strain.
A major driver for the application of microbes in the developed world is the fact
that the public opinion is opposed against the use of agrichemicals. Several supermarket chains have adopted a policy of zero or very low tolerance against chemical
pesticides. Also politicians support this policy, at least in theory. In practice the
process to register biological products in the EU is difficult. Here, the products
should be registered separately in every member country and this process lasts from
several months to several years, depending on the country.
Beneficial microbes often have a clearer effect on plants grown under suboptimal conditions than on plants growing under optimal conditions. Suboptimal
conditions include limitation for nitrogen, phosphorous, or iron; elevated salinity;
and water disbalance. Such conditions are often accompanied by plant diseases. An
example is cucumber grown in salinated and naturally infested soil in Uzbekistan.
Here, as many as 17% of the cucumber plants were diseased by the indigenous
pathogen Fusarium solani. Several European biocontrol strains were very effective
in reducing the percentage of diseased plants.
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The area of salinated soil will increase, for example, at places with intensive
irrigation. Also the predicted climate change will lead to an increase of the area of
salinated agricultural soil, especially in the poorer areas in the world. Here, the
yields are often low and chemicals are therefore too expensive. It can be expected
that the need for the application of the relatively cheap beneficial microbes will
largely increase in these areas. Therefore, it is comforting to know that many
beneficial microbe isolates from plants growing on nonsalinated soil are salt
tolerant and that they can be successfully applied in salinated soil and also in a
warm climate, despite the fact that they have been isolated from a cold climate and
from other plants than cucumber (Egamberdieva et al., submitted). This strong
beneficial effect of bacterization can be explained by the low level of endogenous
microbes in this salinated soil (Egamberdieva et al. 2008), which therefore has a
low buffering capacity against pathogens.
Nowadays many African, Asian, and South American countries with limiting
financial resources for chemical fertilizers and pesticides clearly demonstrate their
determination to use more microbial products as alternatives. Development of
“cottage” type small production units helps local farmers to improve plant’s health
and productivity (Harman et al. 2010). This low tech approach, as well as the increase
in traditional sales of high-tech microbial products, indicates that the use of beneficial
microbes in agriculture will play a more prominent role in the near future. The
development of “cottage” type small production units is a clear example of how
modern knowledge of general biology and of production and use of beneficial
microbes, mainly collected in universities and biocontrol industries in developed
countries, can contribute directly to sustainable agricultural practice and environmental
protection in the economically less developed part of the world.
Acknowledgments Clara Pliego thanks MEC, grant numbers AGL-2005-06347-C03-01,
AGL2008-0543-C02-01 and Junta de Andalucia, Grupo PAI CVI264. Ben Lugtenberg thanks
Leiden University, The European Commission, INTAS, the NWO departments of ALW, CW,
STW as well as the Netherlands (NWO) – Russian Center of Excellence for support. All of us want
to express our sincere gratitude to Prof. Fernando Pliego Alfaro for critical reading and helpful
comments on the manuscript.
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