Table Of ContentNew Perspectives and Approaches in Plant Growth-Promoting
Rhizobacteria Research
New Perspectives and Approaches in Plant Growth-Promoting
Rhizobacteria Research
Editedby:
P.A.H.M. Bakker, J.M. Raaijmakers, G. Bloemberg, M. Hofte, P.Lemanceau
and B.M. Cooke
Reprinted from European Journal ofPlant Pathology, Volume 119Issue 3, 2007
~
Springer
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ISBN 978-1-4020-6775-4
ISSN 978-1-4020-6776-1
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Printed on acid-freepaper
Coverphotos:
From top to bottom: Confocal laser scanning microscopy analyses ofcolonies ofPseudomonasfluorescens
WCS365 marked with green and cyan fluorescent proteins; Model ofthe transcriptional regulation ofACC
deaminase expression in Pseudomonas putida UW4; Overview ofinteractions between biocontrol strains,
plants, pathogens, predators, cooperators, and soil; Circular representation ofthe genome ofPseudomonas
flu0rescens Pf-5
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European Journal of Plant Pathology
Volume 119 . Number 3 . November 2007
Special Issue: Newperspectives and approaches in Plant Growth-Promoting Rhizobacteria research
Edited by:P.A.H.M.Bakker · J.M. Raaijmakers · G. Bloemberg · M. Hdfte •P.Lemanceau · B.M.Cooke
Foreword 241 Dialogues of root-colonizing biocontrol
pseudomonads
Plant responses to plant growth-promoting
C. Dubuis .C. Keel· D. Haas 311
rhizobacteria
L.C. van Loon 243 Promotion ofplant growth by ACC
deaminase-producing soilbacteria
Management of resident plant growth-promoting
B.R. Glick· Z. Cheng' 1.Czarny· 1.Duan 329
rhizobacteria with the cropping system: a review
ofexperience in the USPacific Northwest Effects of plant growth-promoting rhizobacteria
R.J. Cook 255 on nodulation ofPhaseolus vulgaris are
dependent on plant P nutrition
Genomic analysis of antifungal metabolite
R. Remans .A. Croonenborghs .R.T.Gutierrez .
production by Pseudomonasjluorescens Pf-5
J. Michiels' J. Vanderleyden 341
J.E. Loper· H. Gross 265
Quorum sensing as a target for developing control
The magic and menace of metagenomics:
strategies for the plant pathogen Pectobacterium
prospects for the study ofplant
D. Faure· Y.Dessaux 353
growth-promoting rhizobacteria
1.H.J.Leveau 279
Microscopic analysis ofplant-bacterium
interactions using auto fluorescent proteins
Instructions for Authors for Eur J Plant Pathol are available at
G.V.Bloemberg 301 littp://www.springer.com/l0658.
~Springer
Eur JPlant Pathol (2007)119:241-242
DOl 10.1007/s10658-007-9114-z
FOREWORD
Foreword
Peter A. H. M. Bakker' Jos M. Raaijmakers . Guido V. Bloemberg :
Mouica Hofte . Philippe Lemanceau . Mike Cooke
Received:15February2007/Accepted:15February2007/Published online:23March 2007
© KNPV 2007
New perspectives and approaches in plant include Acinetobacter, Agrobacterium, Arthrob
growth-promoting rhizobacteria research acter, Azospirillum, Bacillus, Bradyrhizobium,
Frankia, Pseudomonas, Rhizobium, Serratia,
Plant growth-promoting rhizobacteria (PGPR) Thiobacillus and others. To date, probably the
are defined as root-colonizing bacteria that exert most widely used PGPR in agriculture are
beneficial effects on plant growth and develop Rhizobium and Bradyrhizobium species for their
ment. Root colonization comprises the ability of nitrogen-fixingcapacity in roots of Leguminosae.
bacteria to establish on or in the plant root, to In addition to the promotion of plant growth,
propagate,surviveand dispersealongthe growing PGPR are also employed for controlling plant
root inthe presence of the indigenous microflora. pathogens, enhancing efficiencyoffertilizers, and
Rhizobacteria are considered as efficient micro degrading xenobiotic compounds (rhizoremedia
bialcompetitorsinthe root zone. Representatives tion). The application of PGPR is a growing
of many different bacterial genera have been market.
commercialized and/or introduced into soils,onto There is an active and growing group of
seeds,roots, tubers or other planting materials to scientists working on fundamental and applied
improve crop growth. These bacterial genera aspects of PGPR. Since the late eighties,
P.A. H. M.Bakker M.Hone
Faculty of Biology,Section Phytopathology,Utrecht Pytopathology Lab,Faculty of Bioscience
University,PO Box80084,TB Utrecht 3508,The Engineering, UniversityGhent,Coupure Links 653,
Netherlands Ghent B-90oo,Belgium
e-mail:[email protected] e-mail:[email protected]
J.M.Raaijmakers P.Lemanceau (1:81) .M.Cooke
LaboratoryofPhytopathology,Wageningen UMR 1229Microbiologie du Solet de
University,Binnenhaven 5,PD Wageningen,The l'Environnement, INRA,Universite de Bourgogne,
Netherlands CMSE,17rueSully,BP86510,Dijon Cedex F-21065,
e-mail:[email protected] France
e-mail:[email protected]
G.V.Bloemberg
Institute of Biology,ClusiusLaboratory,Leiden
University,Wassenaarsewegc64,AL Leiden 2333,
The Netherlands
e-mail:[email protected]
~Springer
242 EurJ PlantPathol (2007)119:241-242
developments of PGPR research have been • production, formulation and delivery strate
addressed at International Workshops on PGPR. gies of PGPR
The first meeting was held in 1987 in Orilla, • performance of PGPR in greenhouse trials
Ontario, Canada, and since then in Interlaken, and agricultural fields
Switzerland (1990), Adelaide, Australia (1994), • registration and commercialization of PGPR
Sapporo, Japan (1997), Cordoba, Argentina
In addition to these topics the 7th meeting
(2000) and Calicut, India (2003). In 2006, the
focused on recent developments in genomics,
7th workshop wasorganized in Noordwijkerhout,
proteomics and metabolomics of PGPR. The
The Netherlands, where over 130scientists from
abstract book is available at http://
17 countries worldwide participated and pre
www.bio.uu.nll-fytopath/PDF%20files/ab
sented their results in 49 oral and 69 poster
stract%20book%20PGPR%20final.pdf. Last but
presentations.
surely not least, this meetingwasdedicated to the
Topics addressed during the PGPR workshops
great efforts ofseveral PGPRscientists.These are
include:
Jim Cook, Genevieve Defago, Ben Lugtenberg
• mechanisms of plant growth promotion and and Kees van Loon. In this special issue of the
disease suppression European Journal of Plant Pathology, key con
• traits involved in root colonization by PGPR tributions are published that give an overview of
• the role of PGPR in microbial interactions the work presented at the workshop.
• the molecularand biochemicalbasisofdisease The attendance and excellent contributions by
suppression and root colonization an ever-growing group of young scientists guar
• the role of PGPR in disease-suppressive soils antees a healthy future for PGPR research. Our
• plant responses to PGPR best wishesto DavidWeller and Joyce Loperwho
• discovery of novel PGPR strains and traits will organize the next workshop in the Pacific
• pathogen responses to PGPR Northwest, USA.
• risk assessment of PGPR
~Springer
EurJ Plant Pathol (2007) 119:243-254
DOl 1O.1007/sI0658-007-9165-1
REVIEW PAPER
Plant responses to plant growth-promoting rhizobacteria
L. C. van Loon
Received:4 December2006/Accepted: 3May 2007/Published online:5June 2007
© KNPV 2007
Abstract Non-pathogenicsoilborne microorganisms these signalling pathways. Root-colonizing Pseudo
canpromoteplantgrowth.aswellassuppressdiseases. monasbacteria have been shown to alter plant gene
Plant growth promotion is taken to result from expression in roots and leaves to different extents,
improvednutrientacquisition orhormonal stimulation. indicative of recognition of one or more bacterial
Disease suppression can occur through microbial determinants by specific plant receptors. Conversely,
antagonism or induction of resistance in the plant. plants canalter rootexudation and secrete compounds
Severalrhizobacterialstrainshavebeenshowntoactas that interfere with quorum sensing (QS) regulation in
plant growth-promoting bacteria through both stimu the bacteria. Such two-way signalling resembles the
lationofgrowthandinducedsystemicresistance(ISR), interaction of root-nodulating Rhizobiawith legumes
but it is not clear in how far both mechanisms are and between mycorrhizal fungi and roots of the
connected. Induced resistance is manifested as a majority of plant species. Although ISR-eliciting
reduction of the number of diseased plants or in rhizobacteria can induce typical early defence-related
disease severity upon subsequent infection by a responses in cell suspensions, in plants they do not
pathogen. Such reduced disease susceptibility can be necessarily activate defence-related gene expression.
local or systemic, result from developmental or Instead, they appear to act through priming of
environmental factors and depend on multiple mech effective resistance mechanisms, as reflected by
anisms. The spectrum of diseases to which PGPR earlier and stronger defence reactions once infection
elicited ISR confers enhanced resistance overlaps occurs.
partlywiththatofpathogen-inducedsystemicacquired
resistance (SAR).Both ISR andSAR represent astate Keywords Arabidopsis · Disease suppression .
of enhanced basal resistance of the plant that depends Induced systemic resistance . Plant growth
on the signalling compounds jasmonic acid and promotion . Signal transduction .
salicylic acid, respectively, and pathogens are differ Systemic acquired resistance
entiallysensitivetotheresistancesactivatedbyeachof
Plant growth promotion by rhizobacteria
L. C.van Loon (~)
DepartmentofBiology,Section Phytopathology,Institute Plant roots offer a niche for the proliferation of soil
of Environmental Biology, Faculty of Science, Utrecht
bacteria that thrive on root exudates and lysates.
University, P.O. Box 800.84,3508 TB Utrecht,
Population densities of bacteria in the rhizosphere
The Netherlands
e-mail:[email protected] maybeupto I,OO-fold higher than inbulk soiland up
~Springer
244 Eur JPlant Pathol (2007) 119:243-254
to 15%of the root surface may be covered by micro pathogenic Pythium spp. and other deleterious soil
colonies of a variety of bacterial strains. While these microorganisms through microbial antagonism.
bacteriautilize thenutrients that arereleased from the These observations were the beginning of a research
host for their growth, they also secrete metabolites programme on antagonism between microorganisms
into the rhizosphere. Several ofthese metabolites can that has been continuing to this day at Utrecht
act as signalling compounds that are perceived by University.
neighbouring cells within the same micro-colony, by The stimulation of seed germination and the
cells of other bacteria that are present in the recovery from damping-offof the turfgrass that were
rhizosphere, or by root cells of the host plant (Van caused by the non-pathogenic Pythium spp. were
Loon and Bakker 2003; Bais et al. 2004; Gray and apparent as a promotion of growth relative to
Smith 2005; Kiely et al. 2006). appropriate control plants. However, in reality they
Thebest-studied example ofsignalexchangeisthe were the result of disease suppression. Many bacteria
Rhizobium-legume symbiosis, in which the plant in soil have similar properties (Compant et al. 2005;
releases flavonoid compounds that act as signals for Haas and Defago 2005), but in a number of cases
the bacterium to secrete Nod factors. Nod factors are rhizobacteria can enhance plant growth in the
perceived by plant root hairs and function in a absence of potentially pathogenic microorganisms,
hormone-likefashion toinduce root nodules inwhich as has been shown in e.g. gnotobiotic systems (Van
the Rhizobium bacterium can fix atmospheric nitro Loon and Bakker 2003). Over the years, several
gen. The bacterium grows at the expense of carbo mechanisms ofrhizobacterial growth promotionhave
hydrates from the host, but provides fixed nitrogen been documented (Table 1). The ability to fix
for amino acid biosynthesis in return (Brencic and atmospheric nitrogen is present in various bacterial
Winans 2005; Gray and Smith 2005). This symbiosis species that are either free-living in the soil, or
is a prime example of an intimate relationship associated with plant roots by growing endophytic
between a soil bacterium and its host plant, and ally (Dobbelaere et al. 2003). Poorly soluble inor
illustrates the conceptbehind the term 'plantgrowth ganic nutrients that are rate-limiting for growth can
promoting rhizobacteria' (PGPR): in nitrogen-poor be made available through the solubilizing action of
environments the Rhizobium bacterium promotes bacterial siderophores or the secretion of organic
legume plant growth by providing alimiting nutrient. acids (Vessey 2003).Thehigh populationdensities of
Growth promotion by soil microorganisms is far bacteriain the rhizosphere stimulate nutrient delivery
from uncommon (Glick et al. 1999; Ryu et al. 2005) and uptake by plant roots.
and can be considered part of a continuum in which Other mechanisms of growth promotion involve
interactions between plants and microorganisms modulation of plant regulatory mechanisms through
range from deleterious (pathogens) to beneficial the production of hormones or other compounds that
(PGPR). In the Netherlands, already 75 years ago influence plant development (Frankenberger and
observations were made by an assistant of Professor Arshad 1995). Many bacterial species are capable
Johanna Westerdijk at the Phytopathological Labo of producing auxin and/or ethylene, and synthesis of
ratory 'Willie Commelin Scholten' in Baarn, about gibberellins and cytokinins has also been docu
recovery from damping-off in turfgrass. The person, mented. Introduction of the rhizobacterial strain
by the name of Van Luijk, identified several patho Pseudomonasfluorescens WCS417 inautoclavedsoil
genic Pythium species that were responsible for the promoted growth of Arabidopsis accession Col-Oby
disease, but he also observed that grass seeds 33% (Pieterse and Van Loon 1999). A comparable
germinated to ahigher percentage in non-sterile than growth promotion was seen when Arabidopsis seed
in sterilized soil (Van Luijk 1938). This was the first lings were grown under gnotobiotic conditions on
demonstration in the Netherlands that soil microor vertically oriented agar plates containing half
ganisms can promote plant growth. The reason for strength Hoagland nutrient medium. Compared to
this stimulatory effect of the biological agent present sterile grown control seedlings, WCS417-treated
in the raw soil became clear only later. It turned out seedlings showed enhanced shoot and root develop
that non-pathogenic Pythium spp. were also present, ment, enhanced greening and lateral root formation
took over and counteracted the actions of the (S. van der Ent unpublished observation). Whether
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Eur JPlant Pathol (2007) 119:243-254 245
surface components or secrete compounds that act as
Table 1 Mechanisms of plant growth promotion by rhizo
bacteria 'elicitors' ofplant growth. Plant roots must be able to
perceive and recognize such elicitors in ways similar
Nitrogen fixation totherecognition ofelicitors fromplant pathogens. In
Ion uptake fact,plantpathogens mightinterfere withtheactionof
Iron, zinc, other essential micronutrients PGPR by being perceived by similar receptors.
Phosphate
Production of plant hormones
Auxins, gibberellins, cytokinins, ethylene Plant-mediated disease suppression by
Modulationof plant development rhizobacteria
ACC deaminase
'Elicitors' When plants are growing naturally in soils, one
cannot distinguish whether an apparent growth pro
motion is caused by bacterially stimulated plant
WCS417 produces plant hormones is not known, but growth or through suppression of deleterious soil
promotion of lateral root formation is atypical auxin microorganisms. Non-pathogenic rhizobacteria can
effect (Tanimoto 2005). Obviously, enhanced lateral antagonize pathogens through competition for nutri
root formation increases the capacity to take up ents' production of antibiotics and secretion of lytic
nutrients. For Azospirilium brasilense it has been enzymes (Handelsman and Stabb 1996; Van Loon
shown that auxin is responsible for its growth and Bakker 2003). Such activities are particularly
promoting action in wheat and pearl millet, as important in the rhizosphere where pathogenic fungi
bacterial mutants that had lost 70% of their capacity are attracted to plant roots. However, rhizobacteria
to produce indole-acetic acid had lost their plant canreduce the activity ofpathogenic microorganisms
growth-promoting activity (Barbieri and Galli 1993). not only through microbial antagonism, but also by
Gibberellins and cytokinins both stimulate shoot activating the plant to better defend itself. This
development. Their effects on root growth are less phenomenon, termed 'induced systemic resistance'
well documented. Ethylene is usually considered an (ISR) was firstdescribed by Van Peer et al. (1991) in
inhibitor of plant growth, but at low levels can carnation that was systemically protected against
actually promote growth in several plant species, Fusarium oxysporum f.sp. dianthi upon treatment
including Arabidopsis (Pierik et al. 2006). At moder with strain WCS417, and by Wei et al. (1991) in
ate levels it inhibits both root and shoot elongation, cucumber, where six out of 94 rhizobacterial strains
and at high levels it enhances senescence and organ protected the leaves against anthracnose caused by
abscission (Abeles et al. 1992). The direct precursor Colletotrichum orbiculare. Protection as a result of
of ethylene in the plant biosynthetic pathway, microbial antagonism was excluded because the
1-aminocyclopropane-1-carboxylic acid (ACC) is inducing rhizobacteria and the challenging pathogens
exuded from plant roots together with other amino were inoculated at, and remained confined to,
acids. Rhizobacteria that possess the enzyme ACC spatially separated parts on the same plants. Hence,
deaminase candegrade ACC andutilize itasacarbon the protective effect was plant-mediated.
source. Under such conditions, re-uptake by the roots ISR confers on the plant an enhanced defensive
is prevented and the level of ACC in the roots is capacity (Van Loon et al. 1998; Van Loon and
reduced. As a consequence, ethylene production by Bakker 2005). Upon infection with a challenging
the roots is lowered, relieving inhibition of root pathogen this enhanced defensive capacity is mani
growth. Thus, ACC deaminase-containing rhizobac fested as a reduction in the rate of disease develop
teria can increase root growth by lowering endoge ment, resulting in fewer diseased plants or in lesser
nous ACC levels (Glick 2005). However, bacteria disease severity. The induced resistance is also
lacking ACC deaminase have also been shown to evident locally and sometimes does not extend
increaseplantgrowthandsuchobservations cannotbe systemically (Van Loon 2000). When only local, it
explained by known mechanisms. It is presumed that is difficult to prove, because the inducing bacterium
under such conditions bacterial cells possess certain and the challenging pathogen are not separated from
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246 Eur JPlant Pathol (2007) 119:243-254
each other and direct antagonism is difficult to rule accelerated development leads to enhanced adult
out. Only when specific eliciting components of the plant resistance against late blight caused by Phy
inducer are active in stimulating resistance in the tophthora infestans (Visker et al. 2003).
plant but inactive in antagonizing the pathogen Some reports on ISR have indicated reduced
in vitro on different types of media, can locally symptom expression in the absence of a reduction
induced resistance beinferred. Inductionofresistance in pathogen proliferation. This tolerance of the plant
by live organisms always requires proof that the to the pathogen must have a physiological basis.
organisms cannot contacteach other, aconditionthat Examples are the reduced damage of Pythium
canbe met when aninducing rhizobacteriumremains ultimum-infected cucumbers and lesser extent of soft
confined to the roots and the challenging pathogen rot of potato infected by Erwinia carotovora pv.
colonizes only the leaves. Under such situations the carotovora upon prior treatment of the plants with
inducing bacterium must trigger the roots to locally ACC deaminase-containing rhizobacterial strains. By
produce a signal that moves to the leaves to activate lowering the level of stress ethylene in the plant due
the enhanced defensive capacity systemically. The to pathogenic attack, ACC deaminase acted syner
nature of this mobile signal has so far remained gistically with other mechanisms of biocontrol in
elusive. reducing symptom development without having an
Since its discovery, rhizobacteria-mediated ISR effect on the population density of the pathogen
hasbeen documentedinatleast 15plant species (Van (Wang et al. 2000).
Loon andBakker 2006). Itsinduction hasbeen shown Reduced disease can also be the outcome of
to share several characteristics (Table 2A), but its alterations in the microbial populations in the rhizo
expression can involve different physiological mech sphere as a result of altered host physiology. Num
anisms (Table 2B). ISR can be induced by various bers of resistance-inducing bacteria may be changed,
non-pathogenic microorganisms and by some types or their expression of resistance-inducing traits may
of stress that activate the same response in the plant. be altered (Mark et al. 2005). Plants commonly react
In contrast to R-gene-mediated resistance, it is not to root colonization by rhizobacteria by increasing
specific but active against all types of pathogens, as the release ofexudates, andquantity and composition
well as against several nematodes and insects. Once of root exudates vary with plant developmental stage
induced, plants may remain protected for a consid (Phillips et al. 2004). Thus, plant growth promotion
erable part of their lifetime, indicating that when the could alter root exudation. Moreover, rhizobacteria
stateofISRhasbeen triggered intheplant, itisrather that act as minor pathogens or are perceived by the
stable (Van Loon et al. 1998). plant asapotential threat, arelikely tochange therate
Upon challenge inoculation, ISR is expressed as a and composition of exudates, and to increase the
result of the altered physiological state of the plant. release of lysates.
Expression may take different forms, depending on The population densities and the diversity of the
the activity of the inducing rhizobacterium and the root microfloramay affect the number and activity of
nature of the interaction between the pathogen and resistance-inducing rhizobacteria. Quorum sensing
the plant (Chester 1933).In fact, 'inducedresistance' (QS) within and between bacterial populations is a
is an operational term to denote a condition in which major regulatory mechanism in bacteria to adjust
a plant becomes less diseased compared to a control their metabolism to crowded conditions or other
plant that was not induced. There are many ways in changes in the biotic and abiotic environment
which developmental and environmental factors can (Whitehead et al. 2001). Interference with bacterial
influence plant-pathogen interactions. Damping-off QS by host plants has been documented. Plants can
duetoinfectionbyPythium, Fusarium orRhizoctonia produce and secrete various compounds that mimic
isoften confined to the seedling stage. Any condition QS signals of bacteria and, thereby, alter bacterial
that results in more rapid plant growth will shorten activities in the rhizosphere (Bauer and Mathesius
the vulnerable stage and be apparent as enhanced 2004). The ecological diversity and its consequences
resistance. Rhizobacteria acting through growth pro formetabolic activity ofrhizospherebacteriaareonly
motion could protect plants through this mechanism. poorly known at present and deserve further inves
A similar type of ISR could occur in potato where tigation.
~ Springer
