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Applied Soil Ecology 73 (2014) 1– 8

Contents lists available at ScienceDirect

Applied Soil Ecology

journa l h om epage: www.elsev ier .com/ locate /apsoi l

creening of plant growth-promoting rhizobacteria as elicitor ofystemic resistance against gray leaf spot disease in pepper

in-Soo Sona, Marilyn Sumayoa, Ye-Ji Hwanga, Byung-Soo Kimb, Sa-Youl Ghima,∗

School of Life Sciences and Biotechnology, Institute for Microorganisms, Kyungpook National University, Daegu 702-701, Republic of KoreaDepartment of Horticulture, Kyungpook National University, Daegu 702-701, Republic of Korea

r t i c l e i n f o

rticle history:eceived 4 April 2013eceived in revised form 21 June 2013ccepted 31 July 2013

eywords:okdolant growth-promoting rhizobacteriaPGPR)

a b s t r a c t

This study investigated the effects of plant growth-promoting rhizobacteria (PGPR) isolated from DokdoIsland for growth promotion of pepper and biological control activity against a gray leaf spot diseasepathogen, Stemphylium lycopersici. Screening of PGPR was carried out in the rhizosphere of wild plantElymus tsukushiensis from Dokdo. Rhizobacterial isolates were partially identified based on analysis of 16SrDNA sequences. Phylogenetic analysis was performed using sequences of bacterial isolates for compar-ative purposes. To select PGPR, all bacterial isolates were tested for phosphate solubilization, productionof indole-acetic acid (IAA), and siderophores. Isolates positive for all three characteristics were selectedand tested for growth promotion of pepper as well as potential biological control of S. lycopercisi. All

ystemic acquired resistance (SAR)ray leaf spot diseaseepper

selected isolates were able to enhance plant growth with Kluyvera cryocrescens KUDC1771 showing thehighest plant growth promoting activity. Among selected isolates, four significantly decreased gray leafspot disease severity with Brevibacterium iodinum KUDC1716 providing the highest disease suppression.Moreover, KUDC1716 enhanced expression of pathogenesis-related (PR) protein genes including CaPR4and CaChi2 in the absence of pathogen. These results suggest that B. iodinum induce defense responseagainst S. lycopersici and can be used as a potential agent for biological control.

. Introduction

Dokdo is a volcanic island located in the northeastern part oflleungdo, which is the easternmost region of South Korea. This

egion contains approximately 89 small islands and rocks, includ-ng Dongdo and Seodo Islands. Dokdo Island has disadvantageousonditions for plant growth such as drought, strong winds, steepnclinations, soil salinity, high uric acid concentration, and lack ofrganic nutritive elements. Despite the harsh environment for planturvival, Dokdo contains a healthy plant flora, including 61 speciesCultural Heritage Administration of Korea, 2009a,b). Among theselants, Elymus tsukushiensis, one of the dominant plant commu-ities of Dokdo, displays healthy plant-microbe interactions withlant growth-promoting rhizobacteria (PGPR) (Ham et al., 2009;

eon et al., 2009).PGPR are free-living bacteria that colonize the rhizosphere with

eneficial effects on plant health. PGPR can stimulate plant growth,rotect plants from pathogen infection, and reduce the effects of

biotic stresses, leading to increased crop yields (Babalola, 2010).nhanced plant growth by PGPR can be attributed to the pro-uction of plant growth-promoting hormones, including auxin,

∗ Corresponding author. Tel.: +82 53 950 5374; fax: +82 53 955 5522.E-mail address: ghimsa@knu.ac.kr (S.-Y. Ghim).

929-1393/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsoil.2013.07.016

© 2013 Elsevier B.V. All rights reserved.

gibberellin, and cytokinin, as well as the increased availability oflimited nutrients such as phosphorous, iron, nitrogen, vitamins,and amino acids (Lugtenberg and Kamilova, 2009; Ryu et al., 2003).Among phytohormones, IAA is the primary hormone producedby PGPR and is known as the major auxin in plants. Further, IAAis widespread in the whole body of plants, especially in meris-tematic tissue and growing regions, including germinating seeds;tip of the stem or root and in terminal buds. In addition, IAAelicits various important physiological processes such as divisionof vascular bundles, tropistic responses, and development of lat-eral buds, flower, and fruit. Phosphate solubilization by PGPR isone of the most important factors in plant growth promotion.Phosphorous is considered one of the most essential elementsfor plant growth and development as it participates in energymetabolism and is an important component of nucleic acids suchas ATP and ADP in plants (Taiz and Zeiger, 2003). In fact, largeamounts of phosphate are present in farmland due to chemical fer-tilizers (Belimov et al., 1995; Vessey, 2003). However, phosphatesin soil are rapidly converted to insoluble form upon combiningwith iron (Fe3+), aluminum (Al3+), or calcium ion (Ca3+) (Paul andClark, 1989), resulting in soluble phosphorous for plant use. In

addition, absorption of limited mineral by PGPR plays an impor-tant role in plant growth. Especially, siderophores produced aresecondary metabolites in soil with high affinity iron chelating com-pound secreted by PGPR. Even though iron is an essential nutrient

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J.-S. Son et al. / Appl

f plants, it is relatively insoluble in soil (Vessey, 2003). Therefore,iderophore produced by PGPR play an important role to supplyron in plant.

PGPR possess various direct and indirect mechanisms thatelp to suppress phytopathogens (Kloepper, 1993; Lugtenberg andamilova, 2009; Babalola, 2010). Direct mechanisms involve pro-uction of antibiotics, siderophores, and lytic enzymes such aslucanase and chitinase (Kloepper et al., 1988; Slininger. et al.,004; Kim and Kim , 2008). On the other hand, indirect mech-nisms include induction of local or systemic resistance againstide array of viral, bacterial, and fungal pathogens (Ham et al.,

009; Phi et al., 2010; Son et al., 2012). PGPR also possess vari-us inducible mechanisms for defense against biotic stress. Theseechanisms can be triggered by chemical agents in addition to

virulent, incompatible, or virulent pathogens. Most commonly,nduced resistance in plants is systemic as defense capacity isnhanced in both non-infected as well as primary infected tis-ues. Thus, induction of pathogen resistance in plants is a statef enhanced defensive capacity resulting from proper stimulationy diverse agents such as rhizobacteria (Van Loon et al., 1998).enerally, there are two kinds of defense mechanisms, systemiccquired resistance (SAR) and induced systemic resistance (ISR)Van Loon et al., 1998). SAR is activated by pathogens, resultingn limited infection such as hypersensitive reactions (Choi andwang, 2011). On the other hand, ISR confers enhanced defen-

ive capacity to whole plants not only at sites of rhizobacteriaolonization, resulting in a reduced rate of disease development,ewer diseased plants, and lower disease severity (Zehnder et al.,001). Both SAR and ISR are regulated by distinct signaling path-ays. Specifically, SAR involves accumulation of salicylic acid and

xpression of pathogenesis-related (PR) protein genes, whereas ISRs not always accompanied by activation of PR protein expres-ion (Van Loon, 2007). In addition, 1,3-glucanase and chitinasere among those PR genes expressed for hydrolysis of fungal cellalls. Production of PR proteins is important as they enhance over-

ll resistance in whole plants against diverse pathogens (Adriennend Barbara, 2006). ISR is further potentiated by PGPR and reliesn pathways regulated by jasmonate and ethylene (Bakker et al.,003).

Gray leaf spot disease in pepper seedlings was first reported byinclair et al. (1958) and Blazquez (1969). This disease occurs mostften in mountainous areas exposed low temperatures at nightCho et al., 2001). The main pathogens of gray leaf spot diseasere Stemphylium lycopersici and S. solani, and symptoms includepots no greater than 3 cm in diameter spots with dry, white, andunken centers. The disease develops by interrupting photosynthe-is, resulting in numerous tiny brown to yellowish leaf spots andevere defoliation (Kim et al., 2004). Although chemical fungicidesave been used, no formal chemical or biological agent has beenhown to be effective. Further, chemical agents have toxic effectsn human and animal health by acting as ecocides, resulting in sec-ndary pollution of the environment (Lee, 1997). Further, currentrends encourage the production of organic vegetables and foodsor improved personal well-being. Therefore, use of PGPR as biolog-cal agents could be an effective environment-friendly alternativen the agriculture industry (Shen et al., 2005; Mena-violante andlalde-Portugal, 2007).

Almost all previous PGPR screenings were restrictively car-ied out only under farm-field conditions (Joshi and Bhatt, 2011;arina et al., 2012). However, studies on the identification of PGPRn the rhizosphere of wild plants have not been performed. Theresent study has been carried out to explore the microbial diver-ity of PGPR associated with the rhizosphere of E. tsukushiensis from

okdo Island as well as screen bacterial isolates for their plantrowth-promoting activities and induction of systemic resistancegainst gray leaf spot disease.

l Ecology 73 (2014) 1– 8

2. Materials and methods

2.1. Isolation of microorganisms

All isolates were collected from the rhizosphere of wild E.tsukushiensis from Dokdo Island. To screen rhizobacteria, 1 g of rhi-zosphere soil was mixed with 9 ml of sterilized saline (0.85% NaCl),followed by shaking at 50 rpm for 20 min. Soil suspension was grad-ually diluted and spread on 1/10 tryptic soy agar (TSB, Difco, USA)media, after which plates were incubated at 30 ◦C for 2 days. Bac-terial isolates were characterized based their shape and color andthen isolated as a pure culture. For long-term preservation, all bac-terial isolates were stored in 15% glycerol stocks at −70 ◦C untiluse.

2.2. Cultivation of plants

Pepper (Capsicum annuum L.) seeds (Manita, Nongwoobio,Korea) were surface-sterilized with 1.2% hypochlorite for 30 min,washed 30 min with tap water, and dried at room temperature.Seeds were planted in a 50-hole plastic tray pot (7 cm in diameter)filled with sterilized commercial soil. Pepper plants were grownin a plant growth room under a 12-h light/12-h dark cycle at 25 ◦Cwith 50% humidity. Pepper plants in early 4 leaf stage were used forplant growth promotion assay and induction of resistance againstgray leaf spot disease.

2.3. Incubation of pathogen

S. lycopersici, the causal pathogen of gray leaf spot disease, inocu-lum was prepared as described by Kim et al. (2004). Pathogens weregrown on a V-8 juice agar plate [200 ml of V-8 Juice (Campbell, USA),3 g of CaCO3, 20 g of agar, and 800 ml of distilled water], followedby incubation in a growth chamber under 12 h of fluorescent lightat 20 ◦C and 12 h of dark at 15 ◦C to induce spore formation. Toisolate conidial spores for pepper infection, 7 ml of sterile distilledwater (SDW) was poured into each plate, after which spores weregently rubbed off from the mycelial surface using a plastic loop.Spore suspension was filtered through three-layer cheese cloth andadjusted to 5.0 × 103 spores/ml for induction of systemic resistanceassay. For long-term storage, spores were maintained in 20% sterileglycerol at −70 ◦C.

2.4. Partial identification of bacterial isolates

Bacterial isolates were partially identified by analysis of 16SrDNA sequences. Genomic DNA from isolates was extracted usinga Wizard Genomic DNA Purification Kit (Promega, USA). The 16SrDNA genes were amplified by PCR using the universal sequencingprimer 518F as the forward primer (5′-CCA GCA GCC GCG GTA ATACG-3′) and 800R as the reverse primer (5′-TAC CAG GGT ATC TAATCC-3′) at the Genomic Division, Macrogen Inc., Korea using BigDye(R) Terminator v3.1 Cycle Sequencing Kits (Applied Biosystem,USA). The results of the 16S rDNA sequence analysis were com-pared with registered sequences in the GenBank database usingNCBI Blast server (http://www.ncbi.nlm.nih.gov). The 16S rDNAsequences were deposited in the GenBank database of NCBI.

2.5. Plant growth-promoting characteristics

To identify the beneficial effects of PGPR in plants and select

resistance in pepper, production of indole-3-acetic acid (IAA) andsiderophores was measured, and phosphate solubilization assayswere conducted using all isolates.

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Quantification of IAA production was performed according tohe colorimetric method using Salkowski reagent (50 ml of 35%ClO4, 1 ml of 0.5 M FeCl3) (Glickmann and Dessaux, 1995). Bacte-

ia were incubated with 5 ml of Luria-Bertani (LB) broth mediumithout l-tryptophan, which is a precursor of IAA, at 30 ◦C for 16 hnder rotation at 180 rpm. Then, 20 �l of the bacterial suspensionsdjusted to an optical density of 0.8 was transferred to 5 ml of LBroth media supplemented with 0.1% l-tryptophan. After 16 h of

ncubation, 1 ml of each bacterial suspension was centrifuged at000 × g for 10 min in order to eliminate cells, after which 80 �lf the supernatant was mixed with 160 �l of Salkowski reagent in

96-cell well plate. The mixture was kept in the dark for 25 mino allow color development. After reaction, the absorbance of the

ixture was measured at 540 nm using a Sensident Scan microplateeader (Labsystems, Finland) (Phi et al., 2010). Concentration of IAAn the culture was calculated by based on comparison with an IAAtandard curve.

Siderophore production was measured as described in Schwynnd Neilands (1987). Bacterial isolates were streaked on Chromezurol S (CAS) agar medium divided into equal sectors and incu-ated at 30 ◦C for 2 days.

Solubilization of phosphates was conducted on National Botan-cal Research Institute’s Phosphate (NBRIP) growth agar mediaontaining 10 g of glucose, 5 g of Ca3(PO4), 5 g of MgCl2·6H2O, 0.25 gf MgSO4·7H2O, 0.2 g of KCl, 0.1 g of (NH4)2SO4, 15 g of agar, and000 ml of distilled water (Nautiyal, 1999). Each bacterial isolateas spot-inoculated onto plates at 30 ◦C for 7 days. Clear zones

round the colony indicate capacity to solubilize phosphate.

.6. In vivo plant growth promotion assay

To estimate plant growth promotion, selected bacterial iso-ates were incubated in TSA medium at 30 ◦C for 24 h. Bacteria

ere scraped off the plates, suspended in SDW, and adjusted to09 CFU/ml based on optical density and serial dilution with platesounts. Then, 10 ml of the bacterial suspension per plant waspplied as a soil drench in the rhizosphere of early 3–4 leaf stagelants. The same volume of SDW was used as negative control,hereas 0.5 mM benzothiadiazole (BTH) was applied as a positive

ontrol to trigger SAR (Padidam, 2003). Two weeks after bacterialreatment, growth parameters such as height, fresh weight, and dryeight of both pepper stem and root were measured. Pepper dryeight was obtained after drying in a dry oven at 80 ◦C for 5 days.

he experiment was repeated three times with 10 replications perreatment.

.7. Induction of systemic resistance assay

Induced resistance assay with selected bacterial isolates wasested in early 4 leaf stage pepper plants against the phytopathogen. lycopersici. Bacterial suspension was prepared and applied toepper roots by following the same protocol of the plant growthromotion test. The same volume of SDW was used as a nega-ive control, whereas BTH was used as a positive control. After 1eek, S. solani conidial suspension (adjusted to 5.0 × 103 CFU/ml)as sprayed on abaxial and adaxial leaf surfaces, followed by incu-

ation in a growth chamber under 12 h of fluorescent light at0 ◦C and 12 h of dark at 15 ◦C. Seven days after pathogen chal-

enge, disease severity was evaluated as described by Mehta (1998).ymptoms displayed on each plant were measured on a scale of–9: 0 = no symptoms, 1 = minute white spots, less than 5% diseasedissue; 3 = small white to reddish purple lesions, 5–25% diseased

issue; 5 = dark purple necrotic lesions with chlorosis, 26–50% dis-ased tissue; 7 = necrotic lesions >10 cm long, 50–75% diseasedissue; 9 = lesions coalescing, 76–100% diseased tissue. Diseaseeverity index was calculated according to Falloon et al. (1987). DSI

l Ecology 73 (2014) 1– 8 3

(disease severity index) =∑

(disease category × no. leaves in thatcategory) × 100/(total leaves × 9).

2.8. Reverse transcriptase PCR

To obtain molecular evidence for bacterial-induced expressionof genes related to biotic stress resistance in pepper, we appliedreverse transcriptase polymerase chain reaction (RT-PCR). To ana-lyze the expression of pathogenesis-related (PR) genes, leaves werefirst collected after inoculation with B. iodinum KUDC1716 for 72 h.Leaf tissue samples were ground using a mortar and pestle, afterwhich total RNA was isolated using an RNeasy plant mini kit (Qia-gen, USA) according to the manufacturer’s protocol. Total RNA(1 �g) was employed for first-strand cDNA synthesis using 20 �lof AccuPower RT-Premix (Bioneer, Korea) at 55 ◦C for 1 h and 95 ◦Cfor 5 min. Candidate priming genes were previously described byYang et al. (2009). PCR of priming genes was carried out using thefollowing primers: CaPR1, 5′-ACT TGC AAT TAT GAT CCA CC-3′ and5′-ACT CCA GTT ACT GCA CCA TT-3′; CaPR4, 5′-AAC TGG GAT TTGAGA ACT GCC AGC-3′ and 5′-ATC CAA GGT ACA TAT AGA GCT TCC-3′; CaPR10, 5′-ATG TTG AAG GTG ATG GTG GTG CTG-3′ and 5′-TCCCTT AGA AGA ACT GAT ACA ACC-3′; CaChi2, 5′-ATA TTC CGA ATGTCT AAA GTG GTA C-3′ and 5′-ATT GGA CGA TGG AAG CCA TCA CCAG-3′; CABGLU, 5′-TTT TAG CTA TGC TGG TAA TCC GCG-3′ and 5′-AAA CCA TGA GGA CCA ACA AAA GCG-3′; and CAPO1, 5′-CTA TGGTAT TAG GCC AAG GG-3′ and 5′-GTC ACA AGA ACG GAA TCA CGG-3′. To ensure the equal amounts of RNA, CaActin was analyzed ineach experiment using the following primers 5′-TTG GAC TCT GGTGAT GGT GTG-3′ and 5′-AAC ATG GTT GAG CCA CCA CTG-3′. PCRreactions were conducted using AccuPower PCR premix (Bioneer,Korea) with 100 ng of cDNA and 10 pM of each primer. Amplifica-tion of candidate priming genes was performed on the GeneAmp®

PCR System 2700 (Applied Biosystems) under the following condi-tions: denaturation for 5 min at 95 ◦C, followed by 35 cycles of 30 sat 95 ◦C, 60 s at 55 ◦C, and 45 s at 72 ◦C. PCR products were separatedby 2% agarose gel electrophoresis.

2.9. Statistical analysis

Data from plant growth promotion assay were statistically ana-lyzed by analysis of variance (ANOVA) with treatment meansseparated by Duncan’s multiple range test (DMRT) at p < 0.05 usingSPSS version 19.0 (SPSS Inc., USA).

Data from induction of systemic resistance assay were statisti-cally analyzed by ANOVA, and the treatment means were separatedby least significant difference (LSD) test at p = 0.05 using SPSS ver-sion 19.0 (SPSS Inc., USA).

3. Results

3.1. Bacterial isolation and identification

The rhizosphere of wild E. tsukushiensis from Dongdo of DokdoIsland was studied in order to isolate rhizobacteria and screenPGPR. A total of 126 bacterial isolates were selectively isolatedbased on macroscopic morphological traits. Partial sequencingof 16S rDNA gene sequences resulted in the identification ofisolates grouped into 27 genera (Table 1). Among total iso-lates, Gram-positive isolates predominated (67.46%) with lowG-C content endospore-forming isolates (44.44%), including Bacil-

laceae, Planocaccaceae, and Paenibacillaceae. Gram-negative isolates(32.54%) included 25 gamma-proteobacteria strains, eight strainsof alpha-proteobacteria, seven strains of bacteriodetes, and onlyone strain of beta-proteobacteria (Table 1).

4 J.-S. Son et al. / Applied Soil Ecology 73 (2014) 1– 8

Table 1Identification of bacterial isolates from rhizosphere of E. tsukushiensis based on 16S rRNA sequences.

Gram-negative bacteria Gram-positive bacteria

Generaa Number ofisolates

Genera Number ofisolates

High G-C contents Low G-C contents

Genera Number ofisolates

Genera Number ofisolates

Pseudomonasd 10 Agrobacteriumb 2 Isoptericola 8 Bacillus 43Enterobacterd 6 Rhizobiumb 2 Microbacterium 7 Paenibacillus 8Chryseobacteriume 4 Alcaligenesc 1 Rhodococcus 4 Sporosarcina 5Flavobacteriume 3 Citrobacterd 1 Arthrobacter 3 – –Pantoead 3 Ensiferb 1 Brevibacterium 3 – –Stenotrophomonasd 3 Kluyverad 1 Cellulosimicrobium 2 – –Sphingobiumb 1 Pseudoxanthomonasd 1 Micrococcus 1 – –Sphingomonasb 1 – Gordinia 1 – –

Total 41 29 56

a Identification of bacterial isolates are based on the GenBank database using NCBI database.b Indicates alpha-proteobacteria.

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.2. In vitro screening for plant growth-promoting rhizobacteria

In vitro assays were performed in order to select bacterial iso-ates with plant growth-promoting characteristics. Isolates wereested for siderophore production and phosphate solubilization.ased on the results of these assays, 76% of total isolates were iden-ified as siderophore producers, and 63% were capable of phosphateolubilization (Fig. 1). Despite the stressful and harsh environ-ental conditions, the rhizosphere of E. tsukushiensis contained a

umber of bacterial isolates showing the ability to solubilize phos-hate (Joshi and Bhatt, 2011; Farina et al., 2012). Isolates were alsossayed for production of indole-3-acetic acid (IAA). About 56% ofll isolates produced IAA ranging in concentration from 10 �g/mlo 192 �g/ml (Fig. 1). In the analysis of plant growth-promotingharacteristics, approximately 88% of all bacterial isolates showedt least one plant growth promoting behavior. Fifteen isolates hadhe ability to produce IAA and siderophores, 22 isolates solubilizedhosphate and produced siderophores, and 6 isolates were able toroduce IAA and solubilize phosphate (Fig. 1). About 35% of total

solates were positive for all three characteristics.Based on the results of the plant growth promotion assays,

ine isolates were selected for further experimentation. Theelected isolates were Arthrobacter globiformis KUDC1703,revibacterium iodinum KUDC1716, Bacillus megaterium

UDC1728, Bacillus pumilus KUDC1732, Kluyvera cryocre-cens KUDC1771, Enterobacter ludwigii KUDC1772, Pantoeagglomerans KUDC1793, Pseudomonas putida KUDC1807, andseudoxanthomonas dokdonensis KUDC1809 (Table 2). All selected

able 2dentification and PGP characteristics of selected bacterial isolates.

Strains Gram Nearest neighbora Similarity (%)

KUDC1703 + Arthrobacter globiformis 99

KUDC1716 + Brevibacterium iodinum 99

KUDC1728 + Bacillus megaterium 99

KUDC1732 + Bacillus pumilus 99

KUDC1771 − Kluyvera cryocrescens 97

KUDC1775 − Enterobacter ludwigii 99

KUDC1793 − Pantoea agglomerans 99

KUDC1807 − Pseudomonas putida 99

KUDC1809 − Pseudoxanthomonas dokdonensis 99

a Identification of bacterial isolates are based on the GenBank database using NCBI datab Values indicate the mean of three replicated experiments.c Sequences of the 16S rRNA genes from selected isolates were deposited in the GeneB

Fig. 1. Number of isolates with plant growth-promoting characteristics: phosphatesolubilization, and production of siderophore and indole-3-acetic acid (IAA).

isolates were able to produce IAA, siderophores, and solubilizephosphates.

3.3. Effects of selected isolates on growth of pepper seedlings

The selected bacterial strains were applied to pepper seedlingsfor their ability to promote plant growth. After 3 weeks of

Phosphatesolubilization

Siderophoreproduction

IAA production(�g/ml)b

GenBankaccessionnumberc

+ + 52.30 KC414683+ + 18.89 KC355257+ + 93.33 KC414707+ + 17.78 KC414712+ + 49.15 KC355278+ + 65.11 KC355282+ + 162.48 KC355300+ + 19.33 KC355314+ + 21.56 KC355316

base.

ank database.

J.-S. Son et al. / Applied Soil Ecology 73 (2014) 1– 8 5

Table 3PGP activity of selected isolates according to plant height.

Treatment Plant height (cm)

Root height Total height Increase (%)

(−) Control 13.30 a 23.79 b –BTH 12.87 a 21.28 a –KUDC1703 15.15 cd 26.54 de 11.56KUDC1716 14.89 bc 26.44 de 11.13KUDC1728 14.79 bc 25.27 c 6.22KUDC1732 15.69 d 26.68 ef 12.15KUDC1771 15.64 d 27.45 f 15.38KUDC1775 14.30 b 25.38 c 6.68KUDC1793 15.32 cd 26.80 ef 12.65KUDC1807 15.80 d 26.98 ef 13.41KUDC1809 14.60 bc 25.81 cd 8.49

Values indicate the mean of five replicated experiments. A set of 10 plants was testedper treatment.Ma

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Table 5Induction of systemic resistance by selected isolates against S. lycopersici.

Treatment Disease suppression

Disease severity indexa Reduction (%)b

(−) Control 60.37 b –BTH 27.78 a 54.40KUDC1703 57.40 b –KUDC1716 36.30 a 39.99KUDC1728 47.41 a 21.47KUDC1732 44.45 a 26.38KUDC1771 68.89 b –KUDC1775 55.56 b –KUDC1793 64.15 b –KUDC1807 47.04 a 22.09KUDC1809 60.74 b –

a Values followed by different letters within a column are significantly higher orlower than the (−) control, respectively (p = 0.05).

b

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ean values of the same letter within each column are not significantly differentccording to Duncan’s multiple range test (p < 0.05).

noculation, pepper seedlings were harvested and growth parame-ers such as total height, total fresh weight, and total dry weightf both stem and root measured. The selected bacterial strainsignificantly enhanced all growth parameters compared with theegative control. Total seedling height was enhanced from 6.22%o 15.38% by the isolates (Table 3). In addition, bacterial isolateslevated total fresh weight of seedlings from 28.65% to 59.42% andotal dry weight from 19.84% to 35.19% (Table 4). Among the eightacterial inoculants, treatment with K. cryocrescens KUDC1771howed the highest plant growth-promoting ability for all growtharameters. On the other hand, plants treated with 0.5 mM BTH,hich elicits systemic resistance, showed reduction of plant growth

ompared with the negative control.

.4. Induction of systemic resistance against gray leaf spot disease

All selected isolates were tested for induction of systemic resis-ance against another major gray leaf spot pathogen, S. lycopersici.mong the nine isolates, KUDC1716, KUDC1728, KUDC1732, andUDC1807 significantly reduced disease symptoms compared with

he negative control. Rates of reduction of symptoms by the foursolates were 39.99% in KUDC1716, 21.47% in KUDC1728, 26.38%n KUDC1732, and 22.09% in KUDC1807 (Table 5). B. iodinumUDC1716 both promoted plant growth and induced systemicesistance in pepper.

Among the four isolates with growth-promoting activities, B.odinum KUDC1716 was applied to RT-PCR in order to evaluatehe expression of the PR genes CaPR1, CaPR4, CaPR10, CABGLU, andaChi2 in pepper plants. Pepper leaves from plants treated only

able 4GP activity of selected isolates according to plant fresh and dry weight.

Treatment Fresh weight (g)

Shoot weight Total weight Increase (

(−) Control 1.31 b 1.41 b

BTH 1.00 a 1.07 a

KUDC1703 1.90 de 2.04 de 44.68

KUDC1716 1.80 cd 1.94 cd 37.59

KUDC1728 1.73 c 1.90 cd 34.75

KUDC1732 1.66 c 1.82 c 29.08

KUDC1771 2.08 f 2.25 f 59.57

KUDC1775 1.68 c 1.82 c 29.08

KUDC1793 1.97 ef 2.13 ef 51.06

KUDC1807 1.70 c 1.85 c 31.21

KUDC1809 1.77 cd 1.92 cd 36.17

alues indicate the mean of five replicated experiments. A set of 10 plants was tested perean values of the same letter within each column are not significantly different accordi

Percentage of disease suppression by each bacterial isolates was compared with(−) control.

with SDW did not express PR genes regardless of treatment (Fig. 2).In addition, expression of CaPR1, CaPR10, CaBGLU, or CaPO1 genewas not observed upon any of the treatments (data not shown).However, both BTH and KUDC1716-treated plants induced expres-sion of CaPR4 and CaChi2 compared with only water-treated plants.

4. Discussion

The increasing importance of beneficial bacteria in agriculturehas resulted in many efforts to isolate and identify bacteria associ-ated with the rhizosphere of plants in order to trace their roles inplant growth promotion and protection against phytopathogens.Examples of these useful strains are Bacillus cereus, B. licheniformis,B. pumilus, B. subtilis, Paenibacillus polymyxa, Pseudomonas fluo-rescens, and P. putida (Gutierrez-Manero et al., 2001; Chakrabortyet al., 2006; Kim and Kim, 2008; Phi et al., 2010). Most of thereported PGPR strains were isolated from crops in farm fieldconditions (Joshi and Bhatt, 2011; Farina et al., 2012). Here, wescreened PGPR in the rhizosphere of wild E. tsukushiensis fromDokdo Island and measured its plant growth promotion and protec-tion activities against phytopathogens. A total of 27 bacterial generacomposed of 126 bacteria were selectively isolated, and phyloge-netic assays were performed. The phylogenetic results revealedthat Gram-positive endospore-forming bacteria were predominantwith 44% Bacillus species, followed by 33% Gram-negative bacteria

and 23% Gram-positive high G-C content bacteria. Endospore-forming bacteria are able to overcome harsh stress conditions byforming endospores (Nicholson et al., 2000). Therefore, due to the

Dry weight (mg)

%) Shoot weight Total weight Increase (%)

23.24 b 27.22 b –19.58 a 22.56 a –29.36 de 34.38 de 26.3028.48 c–e 33.80 de 24.1728.22 cd 33.44 c 22.8527.38 c 32.62 ef 19.1331.18 f 36.80 f 35.1928.34 c–e 33.32 c 22.4129.64 e 34.62 ef 27.1928.28 cd 33.72 ef 23.8828.38 c–e 33.48 cd 22.99

treatment.ng to Duncan’s multiple range test (p < 0.05).

6 J.-S. Son et al. / Applied Soil Ecology 73 (2014) 1– 8

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ig. 2. Transciptional expression of PR genes in pepper plants treated with SDW (nf PR genes. Leaf tissues were harvested 0, 24, 48, and 72 h after each treatment. Amiotechnology, Seoul, Korea).

arsh environment of Dokdo, the predominance of Bacillus speciess expected.

Additionally, the beneficial effects of PGPR are associated withheir mechanism and metabolites. Moreover, certain PGPR possess

ore than one plant growth-promoting mechanism (Belimov et al.,995; Lazarovits and Nowak, 1997; Ham et al., 2009). Bacteriaapable of phosphate solubilization are expected to promote plantrowth by increasing phosphorous uptake (Hameeda et al., 2008).pon examination of their phosphate solubilization capacity, about2% of bacterial isolates were able to solubilize phosphate asbserved in NBRIP agar media. Despite the stressful and harsh envi-onmental conditions of Dokdo, the rhizosphere of E. tsukushiensisontained a higher number of bacterial isolates with phosphateolubilization capacity compared with other plants grown underarm-field conditions (Joshi and Bhatt, 2011; Farina et al., 2012).okdo is famous as a diverse avian habitat with various kinds ofirds, mainly Larus crassirostris and Larus argentatus, inhabitinghe island every breeding season. Therefore, bird excrement andggshell debris have accumulated in the soil of Dokdo, leading tohe accumulation of phosphate (Cultural Heritage Administrationf Korea, 2009a,b). From the results of this study, it is estimated thathizospheric soils of Dokdo contain huge amounts of phosphateolubilizer.

Another important plant growth-promoting trait of PGPR ishe production of siderophores (Loper and Buyer, 1991). In

mineral-limited soil environment, bacterial species produceiderophores using an iron-transport system, resulting in reduc-ion of plant pathogens through inhibition of iron absorption.n the present study, approximately 76% of total isolates wereble to produce siderophores. Dokdo assumedly contains manyiderophore-producing bacteria since its environment is made upf small amounts of mineral sources (Hiifte et al., 1994; Ham et al.,009). Some rhizospheric bacteria produce siderophores, and there

s evidence that a number of plants are able to absorb bacteriale3+–siderophore complexes. However, there remains a disputeegarding the effects of bacterial siderophores on iron nutrientptake in plants. While some researchers believe that the contri-ution of siderophores to the overall iron requirements of plants

s small (Glick, 1995), the vast majority of research on bacterialiderophores in the rhizosphere has found strong biocontrol activ-ties due to competitive effects with plant pathogens (Hiifte et al.,994).

The production of phytohormones by bacteria is one of the mostmportant factors of plant growth promotion. Previous researchas revealed the existence of bacteria producing phytohormonesuch as indole-3-acetic acid (IAA), cytokinin, and gibberellinBarazani and Friendman, 1999; Mehnaz et al., 2001; Timmusknd Wagner, 1999; Gutierrez-Manero et al., 2001; Belimov et al.,995). In the present study, about 55% of bacterial isolates were

ble to produce IAA. Further, approximately 88% of all bacterialsolates possessed more than one plant growth-promoting char-cteristic (Fig. 1). These results presume that despite the harshnvironment of Dokdo, multi-functional PGPR in were able to

e control), 0.5 mM BTH (positive control), and KUDC1716. RT-PCR shows induction products were separated by gel electrophoresis and visualized by Redsafe (iNtRON

promote plant growth through well-established plant-microbeinteractions.

To determine plant growth promotion in pepper, nine bacte-rial isolates were selected for further experiments based on theresults of the PGP characteristics assay. All selected isolates wereable to produce IAA, siderophores, and solubilize phosphates. Theresults showed that all treatments significantly improved plantgrowth based on all parameters. Among the nine isolates, K. cryocre-scens KUDC1771 showed the strongest plant growth-promotingactivity, followed by P. agglomerans KUDC1793 and A. globiformisKUDC1703, despite its reduced IAA production. This suggests thata separate mechanism or interplay of various mechanisms, includ-ing other plant hormones or enzymes such as ACC deaminase, isresponsible for the plant growth-promoting effects of KUDC1771.Therefore, further research should be conducted to clarify theplant growth mechanisms of KUDC1771. In addition, investiga-tion of novel compounds related to plant growth produced byKUDC1771 would assist in the development of biological fertilizersas alternatives to chemical fertilizers.

Interactions between bacteria and plant roots can result inresistance to pathogenic bacteria, fungi, and viruses (Lugtenbergand Kamilova, 2009). This phenomenon is called induction of sys-temic resistance (Kloepper, 1993; Van Loon et al., 1998). Grayleaf spot disease caused by the fungal pathogen S. lycopersicicauses numerous tiny spots on leaves as well as severe defolia-tion of pepper seedlings, leading to crop loss. However, no agentfor the control of gray leaf spot disease has been established.Among the nine selected bacterial isolates, four isolates (B. iodinumKUDC1716, B. megaterium KUDC1728, B. pumilus KUDC1732, andP. putida KUDC1809) were able to suppress gray leaf spot diseasein pepper compared with the negative control. Among the well-known beneficial bacterial species in agriculture, B. megaterium, B.pumilus, and P. putida are well-established biological controls incrops; B. megaterium induces disease resistance in Camellia sinen-sis (Chakraborty et al., 2006), B. pumilus increases defense againstblue mold in tobacco (Zhang et al., 2002), P. putida mediates sup-pression of red rot in sugarcane (Hassan et al., 2011). However,until now, there have been few studies on the biological controlability of Brevibacterium species. The genus of Brevibacterium iswidely spread in various habitats such as soil, milk product, clin-ical specimens, and marine environment (Kati et al., 2010). It isGram-positive, non-branching rods, and non-endospore formingbacteria. In aspect of biological agent in plant, Brevibacterium israrely studied, even B. iodinum RS16 was reported to promote theplant growth and decrease the salt stress (Siddikee et al., 2011). Inthis study, B. iodinum KUDC1716 was first reported as an inducerof systemic resistance. KUDC1716 demonstrated plant growth-promoting activity as well as the ability to induce resistance againstpepper pathogens. Therefore, KUDC1716 has potential as a bio-

logical fertilizer and biological control agent against gray leaf spotdisease.

To deeper examine the effects of B. iodinum KUDC1716, weemployed RT-PCR to obtain molecular evidence of plant-microbe

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nteractions. KUDC1716 induced CaPR4 and CaChi2 in pepperlants. Interestingly, KUDC1716 induced PR genes that werelso induced by the chemical BTH. Systemic resistance involveshe induction of genes through SA-dependent, ET-dependent,nd JA-dependent pathways (Yang et al., 2009). RT-PCR resultshowed that CaPR4, which represents SA-dependent signaling,as activated in plants treated with KUDC1716, suggesting thatUDC1716-elicited resistance follows an SA-dependent pathway

Fig. 2). In addition, KUDC1716 also activated the CaChi2 gene,hich is involved in production of class II chitinase. Chitin is a major

omponent of fungal cell walls, and thus chitinase is often applieds an antifungal agent by suppressing fungal growth. Interestingly,UDC1716 induced PR genes similar to BTH, which is a commonlysed chemical control agent known to elicit SAR (Yi et al., 2012).ccording to Van Loon (2007), SAR increases the production of PRroteins, whereas ISR is not commonly related to PR genes. There-ore, the mechanism of KUDC1716-elicited resistance in peppernvolves up-regulation of CaPR4 and CaChi2, and activation of PRenes prior to pathogen infection suggests that KUDC1716-elicitesistance is a form of SAR.

The PGPR isolates in this study protected pepper from gray leafpot pathogens and promoted plant growth. This advantage overhe chemical inducer BTH implies that PGPR are a better alterna-ive for the purpose of biological control. Moreover, the potentialf B. iodinum KUDC1716 as a novel biological agent against grayeaf spot disease should be further explored. Additional investiga-ions into the SAR determinants including volatile compounds ofUDC1716 along with further gene studies are recommended inrder to understand the mechanisms involved in SAR elicitation.

cknowledgements

This research was supported by the Basic Science Research Pro-ram through the National Research Foundation of Korea (NRF)unded by the Ministry of Education, Science and Technology (No.011-0011565).

eferences

drienne, C.S., Barbara, J.H., 2006. Parallels in fungal pathogenesis on plant andanimal hosts. Eukaryot. Cell. 5, 1941–1949.

abalola, O.O., 2010. Beneficial bacteria of agricultural importance. Biotechnol. Lett.32, 1559–1570.

akker, P.A.H.M., Ran, L.X., Pieterse, C.M.J., Van Loon, L.C., 2003. Understanding theinvolvement of rhizobacteria-mediated induction of systemic resistance in bio-control of plant disease. Can. J. Plant Pathol. 25, 5–9.

arazani, O.Z., Friendman, J., 1999. Is IAA the major root growth factor secreted fromplant-growth-mediating bacteria? J. Chem. Ecol. 25, 2397–2406.

elimov, A.A., Kojemiakov, A.P., Chuvarliyeva, C.V., 1995. Interactions between bar-ley and mixed cultures of nitrogen fixing and phosphate solubilizing bacteria.Plant Soil 173, 29–37.

lazquez, C.H., 1969. Occurrence of gray leaf spot on peppers in Florida. Plant Dis.Rep. 53, 756.

hakraborty, U., Chakraborty, B., Basnet, M., 2006. Plant growth promotion andinduction of resistance in Camellia sinensis by Bacillus megaterium. J. Basic Micro-biol. 46, 186–195.

ho, H.J., Kim, B.S., Hwang, H.S., 2001. Resistance to gray leaf spot in Capsicum pepper.HortScience 36, 752–754.

hoi, H.W., Hwang, B.K., 2011. Systemic acquired resistance of pepper to microbialpathogen. J. Phytopathol. 159, 393–400.

ultural Heritage Administration of Korea, 2009a. Natural Heritage of Korea, Dokdo,first ed. Kyungpook National University Press, Daegu.

ultural Heritage Administration of Korea, 2009b. Natural heritage of Korea, Dokdo.In: Park, J.H., Yang, J.Y., Lee, D.H. (Eds.), Flora of Dokdo. Kyungpook NationalUniversity Press, Daegu, pp. 166–221.

alloon, P.G., Falloon, L.M., Grogan, R.G., 1987. Etiology and epidemiology of Stem-phylium leaf spot and purple spot of asparagus in California. Phytopathology 77,

407–413.

arina, R., Beneduzi, A., Ambrosini, A., de Campos, S.B., Lisboa, B.B., Wendisch, V.,Vargas, L.K., Passaglia, L.M.P., 2012. Diversity of plant growth-promoting rhi-zobacteria communities associated with the stages of canola growth. Appl. SoilEcol. 55, 44–52.

l Ecology 73 (2014) 1– 8 7

Glickmann, E., Dessaux, Y., 1995. A critical examination of the specificity of theSalkowski reagent for indolic compounds produced by phytopathogenic bacte-ria. Appl. Environ. Microbiol. 61, 793–796.

Glick, B.R., 1995. The enhancement of plant growth by free-living bacteria. Can. J.Microbiol. 41, 109–117.

Gutierrez-Manero, F.J., Ramos-Solano, B., Probanza, A., Mehouachi, J., Tadeo, F.R.,Talon, M., 2001. The plant-growth-promoting rhizobacteria Bacillus pumilusand Bacillus licheniformis produce high amounts of physiologically active gib-berellins. Physiol. Plant. 111, 206–211.

Ham, M.S., Park, Y.M., Sung, H.R., Sumayo, M., Ryu, C.M., Park, S.H., Ghim, S.-Y.,2009. Characterization of rhizobacteria isolated from family Solanaceae plantsin Dokdo Island. Kor. J. Microbiol. Biotechnol. 37, 110–117.

Hameeda, B., Harini, G., Rupela, O.P., Wani, S.P., Reddy, G., 2008. Growth promo-tion of maize by phosphate solubilizing bacteria isolated from composts andmacrofauna. Microbiol. Res. 163, 234–242.

Hassan, M.N., Afghan, S., Hafeez, F.Y., 2011. Biological control of red rot in sugarcaneby native pyoluteorin-producing Pseudomonas putida strain NH-50 under fieldconditions and its potential modes of action. Pest Manage. Sci. 67, 1147–1154.

Hiifte, M., Woestyne, M.V., Verstraete, W., 1994. Role of siderophores in plant growthpromotion and plant protection by fluorescent Pseudomonads. In: Manthey, J.A.,Crowley, D.E., Luster, D.G. (Eds.), Biochemistry of Metal Micronutrients in theRhizosphere. Lewis Publishers, Boca Raton, FL, USA, pp. 81–92.

Jeon, S.A., Sung, H.R., Park, Y.M., Park, J.H., Ghim, S.-Y., 2009. Analysis of endospore-forming bacteria of nitrogen-fixing bacteria community isolated from plantsrhizosphere in Dokdo island. Kor. J. Microbiol. Biotechnol. 37, 189–196.

Joshi, P., Bhatt, A.B., 2011. Diversity and function of plant growth promoting rhi-zobacteria associated with wheat rhizosphere in North Himalayan Region. Int.J. Environ. Sci. 1, 1135–1143.

Kloepper, J.W., Hume, D.J., Scher, F.M., Singleton, C., Tipping, B., Laliberte, M., Frauley,K., Kutchaw, T., Simonson, C., Lifshitz, R., Zaleska, I., Lee, L., 1988. Plant growthpromoting rhizobacteria (PGPR) on canola (rapeseed). Plant Dis. 72, 42–46.

Kloepper, J.W., 1993. Plant-growth-promoting rhizobacteria as biological controlagents. In: Metting Jr., F.B. (Ed.), Soil Microbial Ecology. Marcel Dekker Inc., NewYork, pp. 255–273.

Kati, H., Ince, I.A., Demir, I., Demirbag, Z., 2010. Brevibacterium pityocampae sp. nov.,isolated from caterpillars of Thaumetopoea pityocampa (Lepidoptera, Thaume-topoeidae). Int. J. Syst. Evol. Microbiol. 60, 312–316.

Kim, B.S., Yu, S.H., Cho, H.J., Hwang, H.S., 2004. Gray leaf spot in peppers caused byStemphylium solani and S. lycopersici. Plant Pathol. J. 20, 85–91.

Kim, J.T., Kim, S.D., 2008. Suppression of bacterial wilt with Bacillus subtilis SKU48-2strain. Kor. J. Microbiol. Biotechnol. 36, 115–120.

Lazarovits, G., Nowak, J., 1997. Rhizobacteria for improvement of plant growth andestablishment. HortScience 32, 188–192.

Lee, K.S., 1997. Evaluation on the effects of pesticide residues to agroecosystem inKorea. Kor. J. Environ. Agric. 16, 80–93.

Loper, J.E., Buyer, J.S., 1991. Siderophore in microbial interactions on plant surfaces.MPMI 4, 5–13.

Lugtenberg, B., Kamilova, F., 2009. Plant-growth-promoting rhizobacteria. Annu.Rev. Microbiol. 63, 541–556.

Mehta, Y.R., 1998. Severe outbreak of Stemphylium leaf blight a new disease of cottonin Brazil. Plant Dis. 82, 333–336.

Mena-violante, H.G., Olalde-Portugal, V., 2007. Alteration of tomato fruit qualityby root inoculation with plant growth promoting rhizobacteria (PGPR): Bacillussubtilis BEB-13bs. Sci. Hortic. 113, 103–106.

Mehnaz, S., Mirza, M.S., Haurat, J., Bally, R., Normand, P., Bano, A., Malik, K.A., 2001.Isolation and 16S rRNA sequence analysis of the beneficial bacteria from therhizosphere of rice. Can. J. Microbiol. 47, 110–117.

Nautiyal, C.S., 1999. An efficient microbiological growth medium for screeningphosphate-solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265–270.

Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., Setlow, P., 2000. Resistanceof Bacillus endospores to extreme terrestrial and extraterrestrial environments.Microbiol. Mol. Biol. Rev. 64, 548–572.

Padidam, M., 2003. Chemically regulated gene expression in plants. Curr. Opin. PlantBiol. 6, 169–177.

Paul, E.A., Clark, F.E., 1989. Soil Microbiology and Biochemistry. Academic Press, NewYork.

Phi, Q.T., Park, Y.M., Seul, K.J., Ryu, C.M., Park, S.H., Kim, J.G., Ghim, S.Y., 2010. Assess-ment of root-associated Paenibacillus polymyxa groups on growth promotion andinduced systemic resistance in pepper. J. Microbiol. Biotechnol. 20, 1605–1613.

Ryu, C.M., Farag, M.A., Hu, C.H., Reddy, M.S., Wei, H.X., Par, P.W., Kloepper, J.W.,2003. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci.U.S.A. 100, 4927–4932.

Schwyn, B., Neilands, J.B., 1987. University chemical assay for the detection anddetermination of siderophores. Anal. Biochem. 160, 46–52.

Shen, S.S., Choi, O.H., Park, S.H., Kim, C.G., Park, C.S., 2005. Root colonizing and bio-control competency of Serratia plymuthica A21-4 against Phytophthrora Blightof Pepper. Plant Pathol. J. 21, 64–67.

Siddikee, M.A., Glick, B.R., Chauhan, P.S., Yim, W.J., Sa, T., 2011. Enhancementof growth and salt tolerance of red pepper seedlings (Capsicum annuum L.)by regulating stress ethylene synthesis with halotolerant bacteria contain-ing 1-aminocyclopropane-1-carboxylic acid deaminase activity. Plant Physiol.

Biochem. 49, 427–434.

Sinclair, J.B., Horn, N.L., Time, E.C., 1958. Unusual occurrence of certain diseases inLouisiana. Plant Dis. Rep. 42, 984–985.

Slininger., P.J., Burkhead, K.D., Schisler, D.A., 2004. Antifungal and sprout regulatorybioactivities of phenylacetic acid, indole-3-acetic acid, and tyrosol isolated from

8 ied Soi

S

T

T

V

V

Zehnder, G.W., Murphy, J.F., Sikora, E.J., Kloepper, J.W., 2001. Application of rhizobac-teria for induced resistance. Eur. J. Plant Pathol. 107, 39–50.

J.-S. Son et al. / Appl

the potato dry rot suppressive bacterium Enterobacter cloacae S11:T:07. J. Ind.Microbiol. Biotechnol. 31, 517–524.

on, J.S., Sumayo, M., Kang, H.U., Kim, B.S., Kwon, D.K., Ghim, S.-Y., 2012. Inducedsystemic resistance against gray leaf spot in pepper by Enterobacter species iso-lated from family Gramineae plants in Dokdo. Kor. J. Microbiol. Biotechnol. 40,135–143.

aiz, L., Zeiger, E., 2003. Plant Physiology, third ed. Sinauer Associates, Inc., Sunder-land.

immusk, S., Wagner, E.G., 1999. The plant-growth-promoting rhizobacteriumPaenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression:a possible connection between biotic and abiotic stress responses. MPMI 12,

951–959.

an Loon, L.C., Bakker, P.A.H.M., Pieterse, C.M.J., 1998. Systemic resistance inducedby rhizosphere bacteria. Annu. Rev. Phytopathol. 36, 453–483.

an Loon, L.C., 2007. Plant responses to plant growth-promoting rhizobacteria. Eur.J. Plant Pathol. 119, 243–254.

l Ecology 73 (2014) 1– 8

Vessey, J.K., 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil255, 571–586.

Yang, J.W., Yu, S.H., Ryu, C.M., 2009. Priming of defense-related genes confers root-colonizing bacilli-elicited induced systemic resistance in pepper. Plant Pathol. J.25, 389–399.

Yi, H.S., Yang, J.W., Choi, H.K., Ghim, S.-Y., Ryu, C.M., 2012. Benzothiadiazole-elicited defense priming and systemic acquired resistance against bacterialand viral pathogens of pepper under field conditions. Plant Biotechnol. Rep. 6,373–380.

Zhang, S., Reddy, M.S., Kloepper, J.W., 2002. Development of assays for assessinginduced systemic resistance by plant growth-promoting rhizobacteria againstblue mold of tobacco. Biol. Control 23, 79–86.