Chapter 11 Plant Growth-Promoting Bacteria: Fundamentals ... · Plant Growth-Promoting Bacteria: Fundamentals and Exploitation Clara Pliego, Faina Kamilova, and Ben Lugtenberg

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 Mediterranea “La Mayora”, Universidad de Malaga -

Consejo Superior de Investigaciones Cientıficas (IHSM-UMA-CSIC), Area de Genetica,

Universidad de Malaga, Campus de Teatinos s/n, 29071 Malaga, 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

295

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 contrib-

ute 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 nutri-

ent 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 metabo-

lites 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

296 C. Pliego et al.

pathogens by production of antibiotics (Chin-A-Woeng et al. 1998; Keel et al.

1992; Haas and Defago 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 ele-

ments in root exudates required for pathogen stimulation or subsequent root colo-

nization (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 manip-

ulating antagonisms between beneficial microbes and pathogens. To do this suc-

cessfully, 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 interdis-

ciplinary 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 environ-

mental 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 photosyn-

thesis. An excellent overview of exudate compounds is written by Uren (2007).

Organic compounds released by the root include organic acids, sugars and polysac-

charides, amino acids, fatty acids, sterols, vitamins, enzymes, and nucleotides.

Moreover, compounds as diverse as auxins, ethanol, flavonoids, and the osmoprotec-

tant glycinebetaine are secreted. Inmany 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 zearale-

none (Phillips et al. 2004), as well as the growth substrate (Kamilova et al. 2006a;

Lipton et al. 1987), can influence the exudate composition.

11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 297

Extensive studies have been carried out on the exudate composition of gnoto-

biotically 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 Fusariumoxysporum 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 StudyingInteractions 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 increas-

ingly gaining the attention of microbial ecologists and plant pathologists, driven by


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 under-

standing 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 bio-

reporters 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 informa-

tion about their functioning and about the possible successful application of com-

mercial 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).


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 onwhether 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, incombination 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 micro-

colonies 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 epider-

mal 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 colo-

nies 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. chlororaphisPCL1391 have not only been shown to be excellent root colonizers but appeared


http://www.clontech.com

also to be able to colonize the hyphae of the phytopathogenic fungus F. oxysporumf.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 thebiocontrol 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 (Perez-

Jimenez 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

Table 11.1 Microscopic visualization of AFP-tagged Pseudomonas strains in the plant rhizo-

spherea

Pseudomonas strain System Analyzed trait Promoter::

gfp-versionReference

P. fluorescens WCS365 Tomato Localization (Plac: gfp) Bloemberg et al.

(1997)

P. chlororaphis Lotus Localization (PpsbA::gfp) Tombolini et al.

(1997)

P. fluorescensDR54-BN14

Barley Localization and

activity

(PA1/03/04:

gfpmut3b)

Normander et al.

(1999)

P. fluorescens WC5365 Tomato Localization (Plac::rfp; Plac::egfp; Plac::ecfp;Plac::eyfp)

Bloemberg et al.

(2000)

P. fluorescens SBW25 Wheat Metabolic activity

and localization

(PpsbA-gfp luxAB) Unge and Jansson

(2001)

P. putida Barley Activity

Localization

(PrrnBP1::gfp[AGA])

(PA1/03/04:gfpmut3

Ramos et al. (2000);

Boldt et al. (2004)

P. fluorescens F113 Alfalfa Localization (Plac:gfpS65T) Villacieros et al.

(2003)

P. fluorescens WCS365

and P. chlororaphisPCL1391

Tomato Localization (Plac::rfp) Bolwerk et al. (2003)

P. brassicacearum Arabidopsisthaliana

Distribution (PA1/03/04:

RBSIIgfpmut3)

(PA1/03/04:RBSII-

dsred)

Achouak et al. (2004)

P. putida Tomato Localization (PA1/03/04:gfpmut3) G€otz et al. 2006P. fluorescens 32 Wheat Localization (Plac:gfp) Van Bruggen et al.

(2007)

P. fluorescens CHA0 Set of plant

species

Gene expression (PphlA-gfp3)(PprnA-gfp3)

De Werra et al. (2008)

P. pseudoalcaligenesand P. alcaligenes

Avocado Distribution (PA1/03/04:gfp) Pliego et al. (2008)

P. putida PCL1445 Tomato Localization (Ptac:gfp; Ptac:mcherry)

Lagendijk et al. (2010)

aPpsbA, 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 anti-

fungal 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


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: basidio-

mycetes, 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 beentagged with different autofluorescent proteins by co-transformation of two plas-

mids, one of which contained a hygromycin resistance gene and the second an afpgene (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. fluorescensWCS365

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 specifi-

cally 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 compe-

tence for the model strain P. fluorescensWCS365, 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 homolo-

gous recombination. Prior to studying their root colonizing abilities, growth rate


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. fluorescensWCS365 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), DeWeert 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

of LPS

Not identified Dekkers et al. (1998)

Outer membrane integrity Homolog to hrtB Dekkers et al. (1998)

Permeability of outer

membrane

colR/colS required for expression

of wapQDe Weert et al. (2006)

Increased pyrimidine

biosynthesis

pyrR Camacho Carvajal

(2001)

Utilization of exudate

compounds

Homolog of mqo encoding malate

dehidrogenase

Homolog of cis-aconitate hydratase

Lugtenberg et al. (2001)

De Weert and

Bloemberg (2007)

Downregulation of

putrescine uptake

Binding site for regulator protein Kuiper et al. (2001a)

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

components of the TTSS

De Weert et al. (2007)

Adaptation to certain

environments

Homolog of mutY De Weert et al. (2004a)


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 rhizo-

sphere (Rainey 1999; Gal et al. 2003) and to analyze gene expression in P. putidaduring colonization of the maize rhizosphere (Ramos-Gonzalez et al. 2005).

Rhizosphere-induced fusions identified genes with probable functions in nutrient

scavenging, chemotaxis and motility, intracellular metabolism, regulation, secre-

tion, 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 knock-

out. This technique was recently applied in Burkholderia vietnamiensis strain G4 toidentify genetic determinants involved in colonization of the pea rhizosphere and in


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 virulence-

associated 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. fluorescensWCS365 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


(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,


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 (Rodrıguez 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 10�18M. 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-siderophorecomplex 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, isinvolved 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 ofErwinia carotovora byP. fluorescens strains was first describedby 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


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 (See-

Fig. 11.1 for the principle of the enrichment procedure). In this way, strain P. putidaPCL 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 rhizoremedia-

tion. Reports on the ability of several endophytic bacteria to degrade some pollu-

tants (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 polyaro-

matic 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 (Garcıa de Salome et al. 2006). In addition to plants, many rhizosphere


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 Garcıa de Salome et al. (2006).

Fig. 11.1 Enrichment of bacteria which compete efficiently for nutrients and niches. The princi-

ple 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)


N

N

COOH

N

N

C

O

NH2

N

N

OH

CH3

OH

OH

O

CH3

HO

O

H3CHO OH

O

HN

Cl

Cl

HN

OH O

H

H

NNH

HN

N

HN

HN

NH N

H

O O

O

OO

O

O

O

OO

O NH2

H

OH

OH

OH

HO

H

CH3

CH2

CH2

CH2

CH2

CH2

CH3

CH2

CH2

O

O

NH

O

H H C N

CL

Cl

NO2

NH

Phenazine-1-carboxylate

2,4-Diacetylphloroglucinol

Phenazine-1-carboxamide Pyocyanin

Pyoluteorin

Viscosinamide

2-hexyl-5- propyl resorcinol

Hydrogen cyanideAHL structure

Pyrrolnitrin

Fig. 11.2 (Continued)


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 develop-

ment, 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

HN

OH

O

N

NN

N

H

NH

HO

H3C

H3C

H2C CH2

HO

CO

O OH

H

COOH

CH3

CH2

OO OH

CH3CH3

CH3

OH

OH

OH

OH

OH

OH

OH

O

Gibberellins (GA3) Auxins (IAA)

Zeatin

Ethylene

Abscisic acid

D-gluconic acid

Fig. 11.2 Structures of important metabolites which play a role in the interaction between plant-

beneficial bacteria and plants


secreted in the rhizosphere as a precursor (Costacurta and Vanderleyden 1995;

Spaepen et al. 2009). Indeed, Kamilova et al. (2006a) observed that P. fluorescensbiocontrol 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 increas-

ing 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 produc-

tion 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 cellsincrease their IAA production, thereby triggering lateral root and root hair forma-

tion, 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 represen-

tative (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 develop-

ment, 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, Arthro-bacter, Bacillus, Burkholderia, Erwinia, Pantoea agglomerans, Pseudomonas,Rhodospirillum rubrum, Serratia, and Xanthomonas (Garcıa de Salome et al.

2001). The spectrum of cytokinins produced by rhizobacteria is similar to that

produced by the plant (Barea et al. 1976; Garcıa de Salome et al. 2001; Frankenberger

and Arshad 1995), of which isopentenyladenine, trans-zeatin, cis-zeatin, and their


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 promo-

tion has been published. Garcıa 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 (Garcıa de Salome 2000). Garcıa 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 germi-

nation, pollen tube growth, and flowering in rosette plants. Like auxins and cyto-

kinins, 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, Bacillusspecies, Bradyrhizobium japonicum, and Rhizobium phaseoli, secrete them in

the rhizosphere (Frankenberger and Arshad 1995; Gutierrez 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.


11.5.5.1 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 ABA-

producing 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 dor-

mancy. ET is synthesized under biotic stress conditions following infection by patho-

gens, 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-50methylthioadenosine (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-ketobutyrateand 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.


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 antag-

onized by the presence and activities of other organisms they encounter (Haas and

Defago 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 micro-

organisms 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 patho-

gen, i.e., parasitism and predation, production of antibiotics, and signal interfer-

ence. 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 effec-

tive in suppressing plant pathogens and diseases. Most biocontrol strains of Pseu-domonas 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


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 documen-

ted 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 cya-

nide (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 mem-

brane 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 metalloen-

zymes, 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 antibac-

terial and antifungal activities (Haas and Defago 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 de-

represses 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


Keel 2003; Lugtenberg and Bloemberg 2004; Duffy and Defago 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. Pro-

duction and secretion of these enzymes by different microbes can result in biocon-

trol 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 Lysobac-ter 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 play-

ing 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, ofwhich 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 competi-

tive 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. How-

ever, 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 AVO110and 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


root: biocontrol strain AVO110 was observed to colonize the root at preferential

penetration sites for R. necatrix infection (intercellular crevices between neighbor-

ing 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 Arabidopsisthaliana, 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


communication mechanism regulates a variety of physiological processes, including

swarming, swimming and twitching motilities, production of pathogenicity/viru-

lence 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 patho-

genic 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 andsymptoms 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 (DeWeert 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. chlororaphisPCL1391 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).


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), coloniza-

tion 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 nutri-

ents and niches with endogenous microorganisms. Therefore, to gain a better

understanding of how biological control functions in the rhizosphere, the interac-

tion 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 compoundsmay use a variety ofmechanisms 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 coloniza-

tion 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 anti-

bacterial 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. fluorescensWCS365 to inhibit pathogenic activity,

survival, and germination of the pathogen significantly contributes to the reduction


of TFRR after introduction of this bacterium into the plant nutrient solution

(Kamilova et al. 2008). Co-inoculation of the biocontrol strain P. pseudoalcali-genes 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 biocon-

trol 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. fluorescenssp. 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;

Rincon 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

(AIR) synthetase

Arginine-/ornithine-binding

protein

Malic enzyme Carbamoylphosphate

synthetase large subunit

Leucine-,isoleucine-,

valine-binding protein

Succinate-semialdehyde

dehydrogenase

ABC transporter

Probable glyceraldehyde-

3-phosphate dehydrogenase

Outer membrane porins

Ribulose-phosphate-3-epimerase

TransaldolaseaAdapted from Lee and Cooksey (2000) and Ahn et al. (2007)


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 penetra-

tion 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 beenreported. 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


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 Defago (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. Thisinhibition correlates with reduction of the level of the AHL autoinducer N-hexanoyl-L-homoserine lactone. The inhibition in P. chlororaphis strain PCL1391 takes placeat 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 antibiotic-

producing 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 mechan-

isms 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 andPCA, 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 labora-

tory 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 chemi-

cals, 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


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 commercia-

lized? 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 pre-

vious paragraphs, is usually directed toward the discovery of the potential plant-

beneficial 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 condi-

tions. 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


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 applica-

tions in agriculture and horticulture. According to European Commission Directive

2001/36/EC amending Council Directive 1991/414/EEC, concerning the introduc-

tion 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.


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 spore-

forming 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 manu-

facturing 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 proper-

ties 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 expen-

sive than formulation of spores. For example, it has been shown for Pseudomonasby 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


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

type

Pathogen Crop

Bacillus subtilis FZB24b Liquid,

powder,

granule

Fusarium, Rhizoctonia,Pythium

Potato, vegetables,

ornamentals, turf

Bacillus subtilisQST 713b

Powder Botrytis, Sclerotinia,Sphaerotheca macularis

Vegetables,

soft fruits

Bacillus subtilis GB03b Powder Aspergillus, Pythium,Rhizoctonia, Fusarium

Cotton, vegetables,

soybean

Pseudomonaschlororaphis MA 342b

Liquid Fusarium, Septoria,Tilletia

Cereals

Streptomyces K61a,b Powder Fusarium Vegetables

Gliocladium catenulatumstrain J1446a,b

Powder Rhizoctonia, Pythium,Phytophthora, Fusarium,Didymella, Botrytis,Verticillium, Alternaria,Cladosporium,

Helminthosporium,Penicillium, Plicaria

Vegetables,

herbs,

ornamentals,

trees,

shrubs,

turf

Trichoderma harzianumstrain T-22a,b

Powder,

granule

Fusarium, Pythium,Rhizoctonia

Vegetables,

ornamentals, turf

Trichoderma asperellumstrain ICC012a,b

Powder Fusarium, Pythium,Rhizoctonia

Vegetables, turf

Trichoderma atroviridestrain IMI206040a

Powder,

granule

Fusarium, Pythium,Rhizoctonia

Vegetables, turf

Trichoderma gamsii strainICC080a,b

Powder Fusarium, Pythium,Rhizoctonia,Verticillium

Vegetables

Trichoderma polysporumstrain IMI206039a

Powder Botrytis, Chondrostereum Soft fruits,

ornamentals

Pythium oligandruma,b Liquid Alternaria, Botrytis,Fusarium, Sclerotinia

Canola, ornamentals,

vegetables

Verticillium albo-atrumstrain WCS850a,b

Liquid Ophiostoma nova-ulmi Elm

aEU Annex I listed strains (http://ec.europa.eu/sanco_pesticides/public)bUS-EPA registration (http://www.epa.gov/oppbppd1/biopesticides/ingredients)


http://ec.europa.eu/sanco_pesticides/public

http://www.epa.gov/oppbppd1/biopesticides/ingredients

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 micro-

organisms 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 microorgan-

ism 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 environ-

ment, 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 insti-

tutes. 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 micro-

organisms, are analyzed by direct observation of the tested organisms under labora-

tory 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 Rhizoc-tonia solani (Adesina et al. 2009; Garbeva et al. 2004; G€otz et al. 2006; Scherwinskiet al. 2008),E. carotovora, andVerticillium 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.


http://www.epa.gov

http://www.epa.gov

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 exam-

ple, several companies that manufacture products with the same active microor-

ganism(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 microorgan-

ism. 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 com-

pany 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 applica-

tions 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


can provide a sound protection which is preferentially better than, or at least

comparable with, other biological or chemical products (if these exist). Commer-

cialization 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 stan-

dard 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 biocon-

trol products must address not only efficacy and, broadly speaking, safety require-

ments, 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


http://www.globalgap.com

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, how-

ever, 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 plant-

beneficial 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 expen-

sive 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 super-

market 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 subopti-

mal 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 effectivein reducing the percentage of diseased plants.


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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