Systemic resistance induced by rhizosphere bacteria

TL;DR: Rhizobacteria-mediated induced systemic resistance (ISR) is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Abstract: Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Summary

Systemic Acquired Resistance

• All plants possess active defense mechanisms against pathogen attack.
• These mechanisms fail when the plant is infected by a virulent pathogen because the pathogen avoids triggering or suppresses resistance reactions, or evades the effects of activated defenses.

454 VAN LOON, BAKKER & PIETERSE

• Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated (47, 48).
• Induced resistance is not the creation of resistance where there is none, but the activation of latent resistance mechanisms that are expressed upon subsequent, so-called “challenge” inoculation with a pathogen (96).
• A signal that propagates the enhanced defensive capacity throughout the plant in SAR appears to be lacking in LAR.
• Both pathogen- and SA-induced resistance are associated with the induction of several families of PRs.
• Experiments withna G-transformed plants indicate that SA is an essential signaling molecule in SAR induced by necrotizing pathogens.

456 VAN LOON, BAKKER & PIETERSE

• Competition for iron was responsible for protecting carnation against fusarium wilt by WCS417.
• The bacterial strain and fungal pathogen remained spatially separated, indicating that WCS417 protected carnation against Fod by a plant-mediated mechanism.
• Similar results were obtained when radish root tips were treated withP. fluorescensstrains WCS417 or WCS374 and For was inoculated on the root base (55).
• These observations established that selected strains of nonpathogenic rhizobacteria can suppress disease by inducing resistance in plants.
• This induced resistance has been termed “induced systemic resistance” (ISR) (42, 73).

Induced Systemic Resistance as the Mechanism of Disease Suppression

• SAR has been documented in multiple plant species (33), whereas studies of ISR by rhizosphere bacteria have concentrated so far on a few species.
• Notably, no ISR has yet been reported in monocotyledons.
• Because many rhizobacteria triggering ISR can also inhibit growth of a pathogen directly, their capacity to suppress disease may involve more than one mechanism.
• In analyses ofP. aeruginosa7NSK2-induced resistance in bean against gray mold (19), P. fluorescensWCS417-mediated protection of carnation against Fusarium wilt (98) and resistance in cucumber against anthracnose induced by any of six PGPR (104) stems, petioles, cotyledons and/or leaf extracts were free from, or contained at most a negligible quantity of, inducing bacteria, implying involvement of ISR.

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• Horizontally on rock wool cubes with the distal part of the roots on cubes contained in a plastic bag, adjacent to another bag with cubes supporting the proximal part of the root system.
• The roots were laid down through an incision in the bags.
• The distal part of the roots was then treated with a bacterial suspension in talcum emulsion.
• Two to three days later, the proximal part of the root system was inoculated with the fungal pathogen.
• Bacterial colonization of the root remained confined to the distal portion of the root and no fungus was recoverable from this part, demonstrating that the bacteria remained spatially separated from the pathogen for the duration of the experiments.

Criteria for Induction of Systemic Resistance

• In those investigations where rhizobacteria-mediated ISR and pathogen-induced SAR were compared directly (35, 62, 67, 73, 100), the level of disease suppression was similar.
• It can be concluded, therefore, that rhizobacteria-mediated ISR is a generally occurring phenomenon resembling pathogen-induced SAR.
• Even when the inducing organism is shown not to be present at the site of challenge with the pathogen, a metabolite produced by a rhizobacterium could be transported through the plant, inhibiting the pathogen directly.
• A derivative of CHAO that overproduces DAP and pyoluteorin protected tobacco roots significantly better than did the wild type against black root rot, caused byThielaviopsis basicola, but at the same time drastically reduced plant growth.
• Out of six strains initially found to induce systemic resistance in cucumber against anthracnose, five did not inhibit the causal pathogenColletotrichum orbiculare on three culture media (104).

2. Suppression of the induced resistance by a previous application of specific inhibitors, such as actinomycin D (AMD), which affect gene expression of the

• This criterion is difficult to apply to ISR mediated by rhizobacteria because inhibitors of plant metabolism affect many processes besides activation of defense mechanisms.
• Eukaryotic fungi will be perturbed in the same way as plants when inhibitors of DNA-dependent RNA- or protein synthesis are employed.
• Inhibition of prokaryotic protein synthesis affects bacteria, as well as plastids and mitochondria.
• This criterion appears most useful if RNA viruses are the challenging pathogens, because these viral pathogens are insensitive to AMD.

3. Necessity of a time interval between application of the inducer and the

• The plant needs time to reach the induced state.
• This result is puzzling in view of the time typically needed to induce ISR and its maintenance thereafter.
• ISR persisted, supporting the notion that, once induced, systemic resistance in the plant is maintained (63).
• If rhizobacteria-mediated ISR is similar to SAR, ISR should also protect against these different types of organisms.
• Resistance induced byP. fluorescensWCS417 in radish is effective against Fusarium wilt, and reduces necrotic lesion formation caused by the avirulent bacterial pathogenPseudomonas syringaepv. tomato(Pst), and the avirulent fungal leaf pathogensAlternaria brassicicolaand a different isolate ofF. oxysporum.

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• Undecimpunctata, and the striped cucumber beetle,Acalymna vittatum(110, 111).
• This observation is particularly interesting because it shows that induction of systemic resistance by these PGPR is associated with a defined change in plant metabolism.
• In the presence of inducing rhizobacteria that may also have antagonistic properties, local protection as a result of induced resistance is difficult to verify experimentally.
• In the development of SAR, a mobile signal is generated, transported from the site of induction both upward and downward in the plant, and generates the induced state in distant tissues (33, 80).
• No cultivar specificity in root colonization patterns of the two strains was observed, so the failure of the strains to enhance protection of the resistant cultivar could not be explained by poor root colonization (63).

Lipopolysaccharide

• In the systemic protection of carnation against Fusarium wilt byP. fluorescens WCS417, heat-killed bacteria or the purified bacterial outer membrane lipopolysaccharide (LPS) were as effective in inducing resistance as were live bacteria (99).
• This observation indicated that the bacterial LPS acts as a determinant of resistance induction by WCS417 in carnation.
• Also in radish, the bacterial LPS appeared to be the trait responsible for resistance induction (56).

466 VAN LOON, BAKKER & PIETERSE

• Or purified LPS consisting of lipid A–inner core–O-antigenic side chain (OA), were as effective as live bacteria when applied to radish roots.
• Bacterial mutants lacking the O-antigen (OA−), as well as their cell wall extracts, were ineffective.
• The resistance-inducing OA of WCS374 was effective not only on roots, but also when applied to the cotyledons.
• Bacterial LPS contributes to growth and survival of the bacteria in planta by aiding in colonization, creating a favorable micro-environment, acting as a barrier to plant defensive compounds, and by modulating host reactions (72).

Siderophores

• The O-antigenic side chain of LPS was a major determinant of ISR under ironreplete conditions, but under iron-limiting conditions, OA−mutants ofP.
• Strain WCS358 did not induce resistance in radish against Fusarium wilt under low-iron conditions, and did not produce SA in culture.
• WCS374 has the largest capacity to produce SA, so SA production in this strain was studied in detail.
• Different siderophores thus seem to trigger ISR.

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• Medium, it reduced the number of spreading lesions caused byBotr tis cinerea on bean by about one half.
• These results demonstrate that induction of systemic resistance in bean by 7NSK2 is dependent on iron nutritional state, and indicate that specific siderophores function not only in the acquisition of iron by the bacteria, but also in the induction of systemic resistance in the plant.
• Strain CHA400, a Sid− mutant of CHA0, still induced PRs but only partial resistance to TNV, implicating the involvement of the pyoverdin siderophore of CHA0 in the induction of resistance against TNV.
• These contrasting results implicate SA production by 7NSK2 in the induction of systemic resistance in bean and tobacco (19, 20), but not by 90-166 in that induced in cucumber and tobacco (75).
• It is possible that a rhizobacterial strain can induce resistance by different mechanisms, depending on the local conditions in the rhizosphere.

Induced Systemic Resistance is Generally Not Associated with Pathogenesis-Related Proteins

• At least eight of the ten major PRs induced in tobacco in response to pathogens causing hypersensitive necrosis, were found in the intercellular washing fluid (IWF) of leaves of plants grown in autoclaved soil in the presence ofP.
• The SA-dependent resistance induced byP. aeruginosa7NSK2 was apparently not associated with changes in protein composition in the protected leaves (G de Meyer, M Höfte, personal communication).
• Neither root-applied low doses of SA nor rhizobacterial strains triggering ISR induced PRs under either high- or low-iron conditions (34, 35).
• Pst induced high levels of PRs, the rhizobacteria did not, even though the latter induced a level of protection against For at least as high.
• This lack of PR-gene expression in treated radish plants suggests that the mechanism of resistance induction byP. fluorescensWCS374 and WCS417 P1: PSA/ARY P2: PSA/PLB QC: PSA/anil T1: MBL July 1, 1998 5:9 Annual Reviews AR061-20.

Absence of Consistent Alterations in Metabolism of Phytoalexins or Inhibitors

• The enhanced defensive capacity of plants exhibiting ISR might rely on the presence of enhanced levels of compounds that inhibit plant pathogens such as phytoalexins.
• The authors have been unable to link ISR to alterations in inhibitory compounds, electrophoretic protein patterns, or enzyme activities in radish.
• The lipid A–inner core was required for activity but the OA had no role (72).
• Upon inoculation on cotyledons, some accumulation of phytoalexins and phenolics was associated with a slight browning reaction, indicating that the bean plants responded defensively to foliar application of these rhizobacterial species.
• No viral antigen was detected by enzyme-linked immunosorbent assay in any asymptomatic PGPRtreated plants (77), indicating that the plants inoculated with inducing bacteria had become refractory to viral infection.

Structural Alterations

• In radish, ISR against Fusarium wilt was expressed primarily as a reduction in the percentage of diseased plants, apparently resulting from a more frequent failure of the causal pathogen.
• For to reach or colonize the vascular tissue (34).
• Such impediment to fungal ingress might involve cellular alterations in the epidermal and cortical cells that inhibit further colonization.

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• Evidence for such PGPR-induced structural modifications was described recently for pea root tissue (9).
• In nonbacterized roots, the pathogen multiplied abundantly through much of the tissue including the vascular stele.
• Upon challenge inoculation, however, pathogen growth was restricted to the epidermis and the outer cortex.
• The walls of these cells were strengthened at sites of attempted fungal penetration by appositions containing large amounts of callose and phenolic materials, effectively preventing fungal ingress.
• Similar wall appositions and papillae were seen in pea roots treated with the rhizobacteriumP. fluorescens train 63-28R upon challenge inoculation with either Fop orPythium ultimum(7, 8), indicating a general induction of defensive physical barriers to pathogen ingress.

A Novel Signaling Pathway for Induced Systemic Resistance in Arabidopsis

• In recent years, the advantages of usingArabidopsis thalianaas a model for studying the molecular genetics of pathogen-induced SAR have become clear through the identification of mutants blocked at specific signaling steps (21).
• To study ISR against foliar pathogens, seedlings were transplanted into autoclaved potting soil into which rhizobacterial strains were mixed.
• Strain WCS374, effective on radish, did not trigger ISR inArabidopsis, whereas strain WCS358, which did not induce resistance on radish, was almost as effective as WCS417 in triggering ISR in Arabidopsis(100).
• Hence, these three strains showed different specificities in the induction of resistance on radish and onArabidopsis.
• In addition, growth ofP. syringae pv. tomatoin the infected leaves was strongly inhibited (73, 100).

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• Whereas the one incapable of producing SA, WCS358, did induce resistance (100).
• Ethylene is an important signaling compound of plant defense responses (12).
• The ArabidopsisJA response mutantjar1 (87) exhibits wild-type levels of pathogen-induced SAR but fails to express rhizobacteria-mediated ISR (74).
• Neither MeJA, nor ACC induced resistance in thenpr1mutant, confirming their action prior to NPR1 (74).
• The hormones might be required without an increase in their endogenous levels.

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• In the assays used, treatment ofArabidopsisseedlings with inducing rhizobacteria occurred a few days to a few weeks before challenge inoculation, and spatial separation between the rhizobacteria and the challenging pathogen was verified.
• Moreover, ISR inArabidopsiswas effective against different pathogens.
• It has been proposed to restrict the term “ISR” to denote the systemic resistance induced by rhizobacteria that is not dependent on SA signaling and not associated with accumulation of PRs (73, 96).
• Preliminary findings suggest that resistance induced by PGPR can be further boosted by application of SA, suggesting that bacteria do not activate the same spectrum of plant responses as pathogens do.
• SAR is associated with the accumulation of PRs, but these are lacking in ISR.

PERSPECTIVES OF INDUCED SYSTEMIC RESISTANCE FOR DISEASE SUPPRESSION UNDER FIELD CONDITIONS

• Because resistance-inducing PGPR are naturally occurring rhizosphere soil inhabitants, the question can be asked whether plants grown under field conditions P1: PSA/ARY P2: PSA/PLB QC: PSA/anil T1: MBL July 1, 1998 5:9 Annual Reviews AR061-20 RHIZOBACTERIA-INDUCED.
• No studies specifically addressing this point appear to have been conducted.
• PGPR can protect plants from disease under commercial cropping conditions and, in certain cases, disease suppression is attributed to induced resistance.
• It may be speculated that endophytic behavior aids in the induction of resistance, because more plant cells are being contacted by the bacteria than by isolates confined to the rhizosphere (9, 27).
• Once activated, the natural resistance mechanisms of the host maintain an enhanced defensive capacity for prolonged periods and are effective against multiple pathogens.

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• In carnation may be linked to vegetative propagation of this plant, because cuttings used as non-inoculated controls could be taken from root stocks that were inadvertently induced (E Hoffland, SCM Van Wees, unpublished observations).
• To reduce the dependence on chemical crop protectants for disease control in agriculture, biological agents are receiving increasing attention.
• Resistance-inducing rhizobacteria offer an attractive alternative in providing a natural, safe, effective, persistent and durable type of protection.
• Protection based on biological agents is not always reliable and is seldom as effective as chemical treatments.
• Different treatments may be combined, and combinations of biocontrol agents that suppress diseases by complementary mechanisms may further reduce disease losses.

ACKNOWLEDGMENT

• The authors thank J Van den Heuvel for critically reading the manuscript.
• Suppression of soil-borne plant pathogens by fluorescent pseudomonads: mechanisms and prospects.
• Compatibility of systemic acquired resistance and micro- bial biocontrol for suppression of plant disease in a laboratory assay.

480 VAN LOON, BAKKER & PIETERSE

• Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat.
• Activation of systemic acquired disease resistance in plants.

482 VAN LOON, BAKKER & PIETERSE

• Plant defense genes are synergistically induced by ethylene and methyl jasmonate.
• Induced resistance in the biocontrol ofPythium aphanidermatumby Pseudomonaspp. on cucumber.J. Phytopathol.142:51–63.

Figures

Figure 1 Signal-transduction pathways leading to pathogen-induced systemic acquired resistance (SAR) and rhizobacteria-mediated induced systemic resistance (ISR) inArabidopsis thaliana. In plant species other thanArabidopsis, rhizobacterially-produced salicylic acid can trigger the SAR pathway as well as ISR.

Table 2 Bacterial determinants and types of systemic induced resistance

Full text

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Annu. Rev. Phytopathol. 1998. 36:453–83
Copyright
c
°
1998 by Annual Reviews. All rights reserved
SYSTEMIC RESISTANCE INDUCED
BY RHIZOSPHERE BACTERIA
L. C. van Loon, P. A. H. M. Bakker, and C. M. J. Pieterse
Department of Plant Ecology and Evolutionary Biology, Utrecht University, P.O. Box
800.84, 3508 TB Utrecht, The Netherlands; e-mail: L.C.vanloon@bio.uu.nl
KEY WORDS: induced systemic resistance, plant growth-promoting rhizobacteria, salicylic
acid, signaling pathways, systemic acquired resistance
ABSTRACT
Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is
phenotypically similar to pathogen-induced systemic acquired resistance (SAR).
Rhizobacteria-mediatedinducedsystemicresistance(ISR)hasbeendemonstrated
against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber,
radish, tobacco, and tomato under conditions in which the inducing bacteria and
the challenging pathogen remained spatially separated. Bacterial strains differ
in their ability to induce resistance in different plant species, and plants show
variation in the expression of ISR upon induction by specific bacterial strains.
Bacterial determinants of ISR include lipopolysaccharides, siderophores, and
salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through
the SA-dependent SAR pathway, others do not and require jasmonic acid and
ethylene perception by the plant for ISR to develop. No consistent host plant
alterations are associated with the induced state, but upon challenge inoculation,
resistance responses are accelerated and enhanced. ISR is effective under field
conditions and offers a natural mechanism for biological control of plant disease.
INDUCTION OF RESISTANCE IN PLANTS
Systemic Acquired Resistance
All plants possess active defense mechanisms against pathogen attack. These
mechanisms fail when the plant is infected by a virulent pathogen because the
pathogen avoids triggering or suppresses resistance reactions, or evades the ef-
fects of activated defenses. If defense mechanisms are triggered by a stimulus
453
0066-4286/98/0901-0453$08.00

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454 VAN LOON, BAKKER & PIETERSE
prior to infection by a plant pathogen, disease can be reduced. Induced re-
sistance is a state of enhanced defensive capacity developed by a plant when
appropriately stimulated (47, 48). Induced resistance is not the creation of re-
sistance where there is none, but the activation of latent resistance mechanisms
that are expressed upon subsequent, so-called “challenge” inoculation with a
pathogen (96). Induced resistance occurs naturally as a result of limited in-
fection by a pathogen, particularly when the plant develops a hypersensitive
reaction. Although tissue necrosis contributes to the level of induced resis-
tance attained, activation of defense mechanisms that limit a primary infection
appears sufficient to elicit induced resistance (33). Induced resistance can be
triggered by certain chemicals, nonpathogens, avirulent forms of pathogens,
incompatible races of pathogens, or by virulent pathogens under circumstances
where infection is stalled owing to environmental conditions. Generally, in-
duced resistance is systemic, because the defensive capacity is increased not
only in the primary infected plant parts, but also in non-infected, spatially sep-
arated tissues. Because of this systemic character, induced resistance is com-
monly referred to as systemic acquired resistance (SAR) (80, 82,89). However,
induced resistance is not always expressed systemically: Localized acquired
resistance (LAR) occurs when only those tissues exposed to the primary in-
vader become more resistant (79). SAR and LAR are similar in that they are
effective against various types of pathogens. A signal that propagates the en-
hanced defensive capacity throughout the plant in SAR appears to be lacking
in LAR.
SAR is characterized by an accumulation of salicylic acid (SA) and patho-
genesis-relatedproteins (PRs)(38, 82,89,95, 102). Accumulationof SAoccurs
both locally and, at lower levels, systemically, concomitant with the develop-
ment of SAR. Exogenous application of SA also induces SAR in several plant
species (29, 82, 97). Both pathogen- and SA-induced resistance are associ-
ated with the induction of several families of PRs. Induction of PRs is invari-
ably linked to necrotizing infections giving rise to SAR, and has been taken
as a marker of the induced state (38, 95,102). Some of these PRs are β-1,3-
glucanases and chitinases and capable of hydrolyzing fungal cell walls. Other
PRs have more poorly characterized antimicrobial activities or unknown func-
tions. The association of PRs with SAR suggests an important contribution of
these proteins to the increased defensive capacity of induced tissues. Plants
transformed with the nahG gene do not accumulate SA or PRs and do not
develop SAR in response to necrotizing pathogens (22,29). The nahG gene en-
codes salicylate hydroxylase, which converts SA into catechol, a product that
does not induce resistance. Experiments with nahG-transformed plants indi-
cate that SA is an essential signaling molecule in SAR induced by necrotizing
pathogens.

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RHIZOBACTERIA-INDUCED RESISTANCE 455
Although SA may be transported from the primarily infected leaves, it does
notappeartobethe primarylong-distancesignal forsystemicinduction (96). So
far, the nature of the signal has not been established (101), butthe levelofSARis
modulated by ethylene and jasmonic acid (JA) (45,52, 89, 97, 103, 108). These
results suggest that induction and expression of SAR are regulated through an
interplay of several signaling compounds.
Plant-Mediated Disease Suppression by Rhizobacteria
Rhizosphere bacteria are present in large numbers on the root surface, where
nutrients are provided by plant exudates and lysates (66, 81). Certain strains
of rhizosphere bacteria are referred to as plant growth–promoting rhizobacte-
ria (PGPR), because their application can stimulate growth and improve plant
stand under stressful conditions (40, 65). Increased plant productivity results
in large part from the suppression of deleterious micro-organisms and soil-
borne pathogens by PGPR (84). Fluorescent Pseudomonas spp. are among the
most effective rhizosphere bacteria in reducing soil-borne diseases in disease-
suppressive soils (107), where disease incidence is low, despite the presence
of pathogens and environmental conditions conducive to disease occurrence.
These bacteria can antagonize soilborne pathogens through various mecha-
nisms (5, 83). For example, bacterial siderophores inhibit plant pathogens
through competition for iron, antibiotics suppress competing microorganisms,
and chitinases and glucanases lyse microbial cells.
Studies on suppression of Fusarium wilt of carnation and radish, caused by
Fusarium oxysporum f.sp. dianthi (Fod) and F. oxysporum f.sp. raphani (For),
respectively, established competition for iron as the mechanism of disease re-
duction by P. putida strain WCS358 (4,25, 58, 59, 76). Under iron-limiting
conditions in the rhizosphere, WCS358 secretes a pyoverdin-type siderophore
(pseudobactin 358) that chelates the scarcely available ferric ion as a ferric-
siderophore complex that can be transported specifically into the bacterial cell.
Siderophores released by Fod or For under these circumstances are less effi-
cient iron-chelators than pseudobactin 358, so iron available to the pathogens
can become limiting in the presence of WCS358. Due to iron deficiency, fun-
gal spore germination is inhibited and hyphal growth restrained, effectively
lowering the chance that the plants become infected, and reducing disease in-
cidence and severity. The plant, in contrast, does not appear to suffer from
iron shortage (26). A bacterial mutant generated by Tn5 transposon mutagen-
esis and unable to produce pseudobactin 358 (WCS358 Sid
−
) did not reduce
disease incidence (25). A different bacterial strain, Pseudomonas fluorescens
WCS417, was about twice as effective as WCS358 in suppressing fusarium
wilt in carnation. However, a Sid
−
mutant of this strain was as effective as
the wild type in suppressing the disease (28). Clearly, a mechanism other than

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456 VAN LOON, BAKKER & PIETERSE
competition for iron was responsible for protecting carnation against fusarium
wiltby WCS417. When WCS417and Fodwere applied to differentparts of car-
nation plants by treating the roots with the bacterium and introducing the fungus
one week later in the stem by slashing, a similar disease reduction was obtained
(98). The bacterial strain and fungal pathogen remained spatially separated,
indicating that WCS417 protected carnation against Fod by a plant-mediated
mechanism. Heat-killed WCS417 proved as effective in suppressing disease as
were live bacteria (99), confirming that the protective effect is plant-mediated.
Similar results were obtained when radish root tips were treated with P. fluo-
rescens strains WCS417 or WCS374 and For was inoculated on the root base
(55). These observations established that selected strains of nonpathogenic rhi-
zobacteria can suppress disease by inducing resistance in plants. This induced
resistance has been termed “induced systemic resistance” (ISR) (42, 73).
RHIZOBACTERIA-MEDIATED INDUCED
SYSTEMIC RESISTANCE
Induced Systemic Resistance as the Mechanism
of Disease Suppression
SAR has been documented in multiple plant species (33), whereas studies of
ISR by rhizosphere bacteria have concentrated so far on a few species. Notably,
no ISR has yet been reported in monocotyledons. The available data on plant
species in which rhizobacteria-mediated ISR has been reported are summarized
in Table 1.
Common procedures to accomplish induced resistance are pouring a suspen-
sion of the bacteria on, or mixing it with, autoclaved soil; dipping the roots of
seedlings in a bacterial suspension at transplanting; or coating seeds with high
numbersof bacteria before sowing(39). Subsequently, seedlings are challenged
with a pathogen. Because rhizobacteria are present on the roots, systemic pro-
tection against root pathogens must be demonstrated by applying the inducing
bacteria to one part of the root system and the challenging pathogen to another
part, for instance by making use of split-root systems. Testing for protection
against foliar pathogens is easier, because the pathogens are naturally separated
from the rhizobacteria. However, rhizobacteria applied to seeds, or to soil into
which seeds are sown or seedlings are transplanted, can move into the interior
of aerial plant tissues and maintain themselves to some extent on the exterior
of aerial surfaces (44,50).
Because many rhizobacteria triggering ISR can also inhibit growth of a
pathogen directly, their capacity to suppress disease may involve more than
one mechanism. Thus, in order to prove that resistance is induced and that

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RHIZOBACTERIA-INDUCED RESISTANCE 457
it is truly systemic, it must be shown that inducing rhizobacteria are absent
from the site of challenge with the pathogen and that the inducing bacterium
and the challenging pathogen remain spatially separated for the duration of
the experiment. Many studies in which induced resistance is considered as the
mechanism responsible for disease reduction have not specifically addressed
this point, leaving the involvement of other mechanisms open to question. For
example, treatment of bean seeds with P. fluorescens S97 reduced the number
of lesions due to halo blight to 17% of that in nontreated controls (1). Protection
was eliminated when the bacterial suspension was autoclaved, indicating a need
for live bacteria for protection to be achieved. Whether the disease suppres-
sion resulted from antagonism or from ISR is not clear, because absence of the
antagonistic bacteria from the aerial parts of the plants was not checked. Yet,
even though conclusive evidence may be lacking, induced resistance can be
an important consequence of tissue colonization with specific nonpathogenic
bacteria or fungi (37).
In analyses of P. aeruginosa 7NSK2-induced resistance in bean against gray
mold (19), P. fluorescens WCS417-mediated protection of carnation against
Fusarium wilt (98) and resistance in cucumber against anthracnose induced by
any of six PGPR (104) stems, petioles, cotyledons and/or leaf extracts were
free from, or contained at most a negligible quantity of, inducing bacteria,
implying involvement of ISR. When spontaneous rifampicin-resistant mutants
were used for treating seeds, some strains were recovered from inside surface-
disinfested roots. However, none of the inducing strains was found in leaves
used for challenge. Upon injection of cucumber cotyledons P. putida 89B-27
and Serratia marcescens 90-166 multiplied in the tissue but were not recovered
from stems 1 or 2 cm above or below the cotyledons (62). Thus, while some
bacteria that induce systemic resistance colonize internal tissues, they do not
appear to establish themselves on challenged leaves, suggesting that neither
competition nor antibiosis is involved in disease suppression (104,105).
In studies on protection of cucumber against diseases caused by soilborne
fungi (61, 113) split-root assays were used in which the inducing bacteria and
the pathogen were simultaneously inoculated on separate halves of seedling
roots, andthen planted in separate pots. ISRwas expressed as delayed symptom
development, reduceddiseaseseverity,andreduceddiseaseincidencecompared
to nonbacterized controls. Movement of inducing bacteria was monitored by
using a bioluminescent transformant, that was detected with a charge-coupled
device camera. The bacteria showed only limited movement within inoculated
pots and did not migrate to the pots in which the pathogen was inoculated,
demonstrating that the PGPR and pathogen remained spatially separated (61).
ISR in radish against Fusarium wilt was demonstrated in a bioassay in-
volving rock wool wetted with nutrient solution (55). Seedlings were placed

Frequently asked questions

Q1. What have the authors contributed in "Systemic resistance induced by rhizosphere bacteria" ?

In this paper, van LOON et al. demonstrated that rhizobacteria-mediated induced systemic resistance ( ISR ) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. 

Q2. How much root is required for induction of resistance in radish?

A minimal concentration of 105 cfu.g−1 root appears to be required for induction of systemic resistance in, for example, radish (76). 

Q3. What is the role of SA in the induction of PRs?

While strong resistance is induced by the rhizobacteria and their OA−-mutants under low-iron conditions, SA is apparently not produced in sufficient quantities to induce PRs. 

Q4. How long does it take for a plant to develop resistance?

once induced, is often maintained for the lifetime of the plant and although the level of resistance expressed diminishes during plant growth, induced leaves remain resistant up to an advanced stage of senescence (13, 49). 

Q5. In what cultivars did P. fluorescens WCS417 reduce incidence?

In carnation, P. fluorescens WCS417 reduced incidence of Fusarium wilt in the moderately resistant carnation cultivar Pallas and less consistently in the susceptible cultivar Lena. 

Q6. Why were the pseudobactins isolated and applied to radish roots?

Because siderophores are produced by bacteria under these conditions, the pyoverdin-type pseudobactins of three WCS strains were isolated and applied to radish roots. 

Q7. What is the effect of a foliar application of rhizobacteria?

Upon inoculation on cotyledons, some accumulation of phytoalexins and phenolics was associated with a slight browning reaction, indicating that the bean plants responded defensively to foliar application of these rhizobacterial species. 

Q8. How was the movement of the bacteria monitored?

Movement of inducing bacteria was monitored by using a bioluminescent transformant, that was detected with a charge-coupled device camera. 

Q9. Why was it expected that rhizobacteria-mediated ISR would still be?

Because rhizobacteria-mediated ISR was found to be independent of SA and not associated with PRs, it was expected that ISR would still be expressed in this mutant. 

Q10. What is the role of rhizobacteria in the defense of cucumber?

Induction of systemic resistance in cucumber against cucumber beetles (Coleoptera: Chrysomelidae) by plant growth-promoting rhizobacteria. 

Q11. Why is endophytic behavior a factor in induction of resistance?

It may be speculated that endophytic behavior aids in the induction of resistance, because more plant cells are being contacted by the bacteria than by isolates confined to the rhizosphere (9, 27). 

Q12. What is the effect of appositions on the walls of the cells?

The walls of these cells were strengthened at sites of attempted fungal penetration by appositions containing large amounts of callose and phenolic materials, effectively preventing fungal ingress. 

Q13. What is the role of a protein in the inhibition of susceptible and hypersensitive reactions?

The inhibition of susceptible and hypersensitive reactions by protein-lipopolysaccharide complexes from phytopathogenic pseudomonads: relationship to polysaccharide antigenic determinants. 

Q14. What is the role of PRs in the development of SAR?

The association of PRs with SAR suggests an important contribution of these proteins to the increased defensive capacity of induced tissues. 

Q15. Why was the bioassay adapted to Arabidopsis?

To this end, the bioassay for studying ISR in radish was adapted to Arabidopsis in order to study induction of systemic resistance against root pathogens (73). 

Q16. What is the role of ethylene perception in the signal-transduction pathway?

Because WCS417 colonized roots of the etr1 mutant and wild-type to the same extent, these observations implicate ethylene perception as a specific and essential step in the signal-transduction pathway leading to ISR. 

Q17. What is the reason for the lack of expression of ISR in ecotype RLD?

The lack of expression of ISR in ecotype RLD, as well as in the jar1, etr1 and npr1 mutants, rules out the possibility that rhizobacteria-produced antibiotics might have been responsible for, or contributed to, ISR.