Synergistic Plant Growth Promotion by the Indigenous

J. Korean Soc. Appl. Biol. Chem. 52(5), 531-538 (2009) Article

Synergistic Plant Growth Promotion by the Indigenous Auxins-producing PGPR Bacillus subtilis AH18 and Bacillus licheniforims K11

Jong-Hui Lim and Sang-Dal Kim*

Department of Applied Microbiology, College of Natural Resources, Yeungnam University,

Gyeongsan 712-749, Republic of Korea

Received April 27, 2009; Accepted June 5, 2009

In this study, we invested the synergistic plant promotion ability of red-pepper and tomato by the

selected multi-functional PGPR: Bacillus subtilis AH18 and Bacillus licheniformis K11. The both

strains of PGPR B. licheniformis K11 and B. subtilis AH18 produced the auxins, antifungal β-

glucannase, and siderophores, and were capable of solubilizing insoluble phosphates. The auxins

produced by B. subtilis AH18 and B. licheniformis K11 were purified and identified from culture

filtrates using PVP column, Sephadex LH-20 column chromatography, HPLC, GC-MS, and 1H-

NMR. The purified auxinAH18 was confirmed to have derivatives composed with IAA of MW 175,

IBA of MW 203, and IPA of MW 189. The amount ratio of auxinAH18 producing was as follows:

IAA:IBA:IPA=1:1.5:2.6. The purified auxinK11 consisted of an IBA of MW 203. B. licheniformis

K11 and B. subtilis AH18 stimulated seed germination and root growth of red-pepper, tomato,

green onions, and spinach. In particular, red-pepper and tomato plants displayed up to 20%

increased root, stem, and leaf growth. When the pots were simultaneously treated with a

combination of auxinAH18 and auxinK11, the growth rates of red pepper and tomato plants were over

20% greater than observed with treatment with either auxin alone.

Key words: auxins, indole-3-acetic acid, indole-3-butyric acid, PGPR

Rhizobacteria, which inhabit the rhizosphere of various

plants, can stimulate plant growth and disease

suppression directly or indirectly [Loper and Schroth,

1986; Jung et al., 2006a]. PGPR are capable of

synthesizing phytohormone auxins, display antagonistic

activity against phytopathogens, solubilize insoluble

phosphate, and produce ACC deaminase that hydrolyzes

the ACC of the ethylene precursor [Timmusk and

Wagner, 1999; Shimon et al., 2004; Jung et al., 2006b].

These mechanisms can induce mutualism between plants

and the PGPR. Among these, auxin is one of the most

well-known hormones, largely owing to its pivotal

functions in the initial processes of lateral and

adventitious root formation [Gaspar et al., 1996; Idris et

al., 2007] and root elongation [Yang et al., 1993]. As

well, PGPR may also enhance plant auxin synthesis

[Kloepper et al., 2004; Yao et al., 2006].

Auxins are difficult to analyze because they occur in

very low amounts in culture extracts, which are quite rich

in interfering substances [Costacurta and Vanderleyden,

1995; Vandeputte et al., 2005]. Common purification

procedures including column chromatography, solid

phase extraction, and liquid-liquid extraction are used to

purify auxin and indole compounds [Dobrev et al., 2005].

However, these procedures generally require significant

amounts of solvents, time, and labor [Mohammad and

Prasad, 1998; Dobrev et al., 2005]. Mass spectrometric

detection is a well-recognized and specific method that

enables the sensitive and accurate measurement of auxin

and indole compounds in crude extracts [Muller et al.,

2002; Dobrev et al., 2005]. In recent studies, GC-MS

analysis have confirmed gibberellin production by B.

pumilus and B. licheniformis, as well as IAA production

by Pseudomonas fluorescens and Azotobacter chroococcum

[Martinez-Toledo et al., 1998; Leonid et al., 2000;

Gutierrez-Mañero et al., 2001].

Most approaches for plant growth promotion and plant

diseases suppression have used single microbial agents.

*Corresponding authorPhone: +82-53-810-2395; Fax: +82-53-810-4663E-mail: [email protected]

Abbreviations: ACC, 1-aminocyclopropnae-1-carboxlyic acid;EtOAc, ethyle acetate; GC-MS, gas chromatography-mass spec-trometry; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid;IPA, indole-3-propionic; PGPR, Plant growth promoting rhizo-bacteria; PVP, polyvinylpyrrolidone

doi:10.3839/jksabc.2009.090

532 Jong-Hui Lim and Sang-Dal Kim

But, single microbial agents are not likely to be active in

all soil environments in which they are applied. Rather,

the application of a mixture of microbial agents more

closely mimics the natural situation, broadens the

spectrum of biocontrol activity, enhances the efficacy and

reliability of control, promotes the plant growth, and

allows the use of variously combined mechanisms

without the need for genetic engineering [Georg et al.,

1998].

In this study, we described the purification and

identification of auxins and indole compounds produced

by the PGPR strain B. subtilis AH18 and B. licheniformis

K11; the purified auxins were also evaluated to assess

plant growth promotion ability on red-pepper and tomato

plants.

Materials and Methods

Bacteria strain and growth condition. B.

licheniformis K11 and B. subtilis AH18 were isolated

from local field soils in Yeongcheon, Korea [Jung et al.,

2006a; 2006b]. The PGPR bacteria were grown for 3

days in King B medium containing 0.1% tryptophan at

37oC.

Detection of auxin. A colorimetric technique using

Salkowski regent consisting of 27.6 mM FeCl3 and 6.6 M

H2SO4 was used to detect auxins and indole compounds

produced from the selected PGPR strain. Two milliliters

of the reagent were added to 1 mL of each bacterial

culture supernatants and thoroughly mixed in a 3 mL

spectrophotometer cuvette, after which the mixture was

left in the dark for 30 min at room temperature. The

optical density was measured with a spectrophotometer at

535 nm. The standard curve of auxin was determined

using commercial IAA (Sigma-Aldrich, St. Louis, MO)

in a concentration range from 5-45 µg/mL.

Extraction and separation of auxin. The culture

supernatant was adjusted to pH 2.8 using 30% phosphoric

acid and was extracted with an equal volume of ethyl

acetate (EtOAc). The EtOAc layer was washed three

times with a 1/2 volume of ddH2O to remove all

impurities except for indole compounds. The EtOAc was

then evaporated on a rotary evaporator (Bun RE, Buchi,

Frawell, Switzerland) in vacuo at 35oC. The indole

compounds were dissolved in 50% methanol (MeOH).

Purification of auxin. The partially purified indole

compounds produced by B. subtilis AH18 and B.

licheniformis K11 were subjected to PVP column

chromatography (1.5×30 cm), and eluted using a hexane-

EtOAc step-wise gradient of 4:1 (v/v), 1:1, 1:4-1:9. The

eluted fractions showing auxin activity on Salkowski

reagent were reduced to dryness in vacuo, then

resuspended in 50% MeOH. The active fractions were

applied to a 3×70 cm Sephadex LH-20 column, eluted at

0.5 mL/min with 50% MeOH, and the solvent was

removed in vacuo. For preparative-HPLC analysis, indole

compounds were analyzed in a PrepStar 218 Preparative

HPLC System (Varian Inc., Palo Alto, CA) equipped with

a 250×21.4 mm Microsorb 60-8 C18 column (Varian).

The column was eluted with 80% solvent A (1% acetic

acid) and 20% solvent B (100% MeOH) as a mobile

phase at a flow rate of 0.5 mL/min. The indole compounds

were detected at 254-280 nm.

GC-MS and nuclear magnetic resonance (NMR)

analyses. The putative indole compound fractions,

collected from the C18 column, were methylated with

diazomethane (Fig. 1). The methanol and ether were

evaporated with a stream of nitrogen gas in glass vials

with screw-top caps [Guinin et al., 1986]. The samples

were then analyzed at the Korea Basic Science Institute,

Daegu Branch by GC-MS (5973 inert GC/MSD, Agilent

Technologies, Santa Clara, CA). The column utilized was

Fig. 1. Purification scheme of auxins from B. subtilisAH18 and B. licheniformis K11.

Plant growth promotion by auxin-producing PGPR 533

a DB-5 column (30 m×0.225 mm). The GC injector

temperature was 250oC and the oven was programmed

for 2 min at 80oC, 1 min at 10oC, and 10 min at 280oC.1H-NMR analysis was conducted at the Yeungnam

University Instrumental Analysis Center using Fourier

transform-NMR (Varian) operating at 600 MHz.

Adventitious root development in hypocotyl cuttings

of Mung bean. Seed of mung bean (Vigna radiate L.)

were germinated in moist vermiculite and grown for 3

days in a growth chamber under 12 h photoperiod (light

intensity 5,000 lux) with day and night temperatures at

25oC. Cuttings were prepared by removing cotyledon and

cutting off the root system 3 cm below the cotyledon

node. These were placed in a 25 mL vial containing 15

mL of distilled water or test solution. The cuttings were

treated with the purified auxinAH18 and auxinK11 or distilled

water for 24 h prior to transfer to distilled water. The

distilled water was renewed every 24 h. The number and

length of adventitious roots were determined 7 days after

the cuttings were made.

Seed germination. For the seeding bioassay, a variety

of plant seeds including pepper, tomato, spinach, radish,

and green onion were prepared and surface-sterilized by

soaking in 70% ethanol for 1 min and seed disinfection

reagent (sodium hypochlorite: water: 0.05% Triton X-100

in a v/v ratio of 3:2:2) for 5 min. Residual bleach was

removed by rinsing seeds three times in sterile water prior

to storage in a dessicator at 4oC for 3 days. With three

replications for each treatment, approximately 40 seeds

were transferred to Petri dishes containing filter paper

wetted with 1 mL of sterile water, then autoclaved. The

partially purified PGP (plant growth promotion)AH18 and

PGPK11 collected from the EtOAc fraction were resuspended

and treated with 10 mL of sterile water at concentrations

of 10 ppm. The Petri dishes were incubated in darkness at

28oC. The number of germinations of each seed sample

was measured after 3 days. The seeds were also incubated

in 10 mL of sterile water under the same conditions as the

controls.

In vivo pot test of plant growth promotion. Seeds of

red-pepper and tomato were washed with 70% ethanol

and seed disinfection reagent. One seed from each of the

plant was planted and grown in 9×8 cm (diameter×depth)

pots using 200 g of soil TKS 2 (FloraGard Ltd.,

Oldenburg, Germany) until the five-leaf stage at 28oC and

a relative humidity of 50%. The purified auxinAH18 and

auxinK11 were resuspended and treated with 10 mL of

sterile water at concentrations of 10 ppm per pot. The

PGPR strains of 108 cells were washed, resuspended in 10

mL of water, were treated in the pots. The plants were

watered every 5 days with 50 mL of sterile water to

maintain adequate soil moisture. The plants were also

incubated in 10 mL of sterile water under the same

conditions as the controls. Each experiment included 20

plants per treatment with three replications. The growth

promotion activity was calculated from the dry weight

and stem elongation of the plants. The number of leaves

per plant and the leaf sizes were determined.

Fig. 2. GC-MS spectrum of the auxinAH18 and auxinK11 produced by B. subtilis AH18 and B. licheniformis K11. (A),GC-MS spectrum of the purified auxinAH18; (B), GC-MS spectrum of the purified auxinK11. Respective retention times ofIAA, IBA, IPA were 14.56, 15.54, and 16.62 min. The auxinAH18 was composed of an IAA: IBA: IPA ratio of 1:1.5:2.6.The column was a DB-5 column (30 m×0.225 mm). The GC injector temperature was 250 and the oven was programmedfor 2 min at 80oC, 1 min at 10 and 10 min at 280oC.


Results and Discussion

Purification of auxin. The auxin containing indole

compounds produced by B. subtilis AH18 and B.

licheniformis K11 were partially purified by EtOAc

extraction. The partially purified auxinAH18 and auxinK11

dissolved in 50% MeOH were then applied to a PVP

column and subsequently a Sephadex LH-20 column.

The fractions exhibiting auxin activity according to the

Salkowski assay were pooled and applied to a preparative

HPLC. The purified auxinAH18 showed a broad peak at 17-

20 min and purified auxinK11 exhibited a single peak at 18

min. The broad peak of the purified auxinAH18 suggested

that the indole compounds of auxinAH18 were not well-

separated and indole compounds with similar retention

time were contained, as compared to that of the purified

auxinK11. A PVP column chromatography was utilized for

the separation and purification of the EtOAc extract.

Among the fractions collected from the second Sephadex

LH-20 column, 13 fractions (No. 21-33) formed a faint

yellow color and five fractions (No. 29-33) formed an

intense pink color in the Salkowski reaction. The

Salkowski reagent recognizes and reacts specially with

indole compounds to form a pink color in solution, which

can be quantified using a spectrophotometer.

GC-MS and NMR analysis. GC-MS and 1H-NMR

were utilized for the identification of indole compounds

in the HPLC fractions of purified auxinAH18 and auxinK11.

The compounds of the auxinAH18 were identified as IAA

(C11H11O2N, MW 175), IBA (C12H13NO2, MW 203), IPA

(C11H11NO2, MW 189). The auxinK11 was identified as

IBA (C12H13NO2) of MW 203 (Fig. 3-5). The IAA: IBA:

IPA ratio of the auxinAH18 produced by B. subtilis AH18

was 1:1.5:2.6 (Fig. 2).

Adventitious root development in hypocotyl cuttings

of mung bean. Adventitious root formation was visible

at the base of mung bean hypocotyls 3 days after cuttings

were made. When the auxinAH18, auxinK11 and commercial

auxin (IAA and IBA) were compared for their ability to

Fig. 3. 1H-NMR spectrum (600 Hz) of the axuinAH18.(A), IAA spectrum of the purified auxinAH18; (B), IBAspectrum of the purified auxinAH18; (C), IPA spectrum ofthe purified auxinK11.

Fig. 4. 1H-NMR spectrum (600 Hz) of IBA of theauxinK11.


stimulate the formation and development of adventitious

roots at a concentration of 10 ppm, the results showed that

the purified auxinAH18 and auxinK11 were more effective

plant growth promoting hormones than commercial auxin

(Table 1). But, the auxinAH18, auxinK11, IAA and IBA

applied at high concentration, up to 20 ppm, inhibited

adventitious root formation and development (Table 1).

At their optimal concentrations (10 ppm for each), purified

auxinAH18 and auxinK11 induced 16.6 and 14.3 roots per

cutting, respectively, and simultaneously promoted 6.6

and 6.1 mm per root, respectively (Table 1). Also, a

synergistic effect was seen in the formation and

development of adventitious roots when the cuttings were

treated simultaneously with a mixture of purified

auxinAH18 and auxinK11. When both compounds were

present in the mixture at 5 ppm 19.2 roots per cutting and

7.2 mm per root were produced, while separate application

of 10 ppm auxinAH18 and auxinK11 produced 16.6 and 14.3

roots per cutting, respectively, and 6.6 and 6.1 mm per

root, respectively (Table 1). The purified auxinAH18 and

auxinK11 could induce more effective root formation and

development than the commercial auxin mixture at low

concentration, with the mixture of purified compounds

producing a synergistic effect.

Seed germination. The partially purified PGPAH18 and

PGPK11 produced by B. subtilis AH18 and B.

licheniformis K11 stimulated seed germination of red-

pepper, tomato, green onion, spinach, and radish plants.

The germination of seeds treated with the partially

purified PGPAH18 and PGPK11 was 13% higher, on average,

than the seeds from the control seeds treated only with

water (Fig. 6).

In vivo pot test of plant growth promotion. Auxin

produced by PGPR, which is synthesized in minute

amounts, affects many activities in plant growth and

development and is widely used in the agriculture

[Karadeniz et al., 2006]. Additionally, many indole

compounds, including IBA, indole-pyruvic aicd, indole-

acetamide acid, and indole-carboxylic acid, may also

prove to be involved in root formation and seed

germination [Costacurta and Vanderleyden, 1995]. So,

we verified the plant growth promoting activity of auxin

produced by the PGPR strains AH18 and K11 in pepper

and tomato plants.

Fig. 5. Putative chemical structure of the auxinAH18 and auxinK11. (A), IAA (C11H11O2N, MW 175); (B), IBA (C12H13NO2,MW 203); (C), IPA (C11H11NO2, MW 189). The compounds of the auxinAH18 identified the IAA (C11H11O2N, MW 175), IBA(C12H13NO2, MW 203), IPA (C11H11NO2, MW 189) and that of the auxinK11 identified IBA (C12H13NO2, MW 203).

Table 1. Synergistic growth by the mixed treatment of auxinAH18 and auxinK11 on red-pepper and tomato

Dry weight (mg) Stem elongation (cm) Roots (cm)

Red-pepper

Only water 14±1.6 6.3±0.4 4.9±0.4

AuxinAH18 38±2.2 7.4±0.9 5.3±0.2

AuxinK11 24±1.9 8.5±0.7 5.2±0.2

AuxinAH18+AuxinK11 86±2.1 11.6±0.90 8.2±0.4

Tomato

Only water 31±2.3 8.0±0.6 5.3±0.3

AuxinAH18 38±2.6 10.0±0.80 6.5±0.7

AuxinK11 45±2.7 13.3±0.90 6.8±0.7

AuxinAH18+AuxinK11 82±2.6 16.0±0.80 12.3±1.10

Commercial IAA (Sigma-Aldrich) and the purified auxins were treated at concentrations of 100 ppm. The plants were

watered every 5 days with 50 mL of sterile water. After 20 days of treatment, the growth promotion activity was

determined. Values are expressed as the means of three replicates, each containing 20 plants. Standard errors were

determined at p≤0.05.


The purified auxinAH18 and auxinK11 promoted plant

growth, as evidenced by the measurements of dry weight,

stem elongation, and root length (Fig. 7). The average dry

weights of the purified auxinAH18- and auxinK11-treated

plants were 38 and 24 mg (red-pepper), and 28 and 45 mg

(tomato), respectively, when compared with the control

red-pepper (14 cm) and tomato (31 cm) plants treated

only with water (Table 2). The stems and roots of the

purified auxinAH18- and auxinK11-treated plants were 25%

longer than the plants treated only with water (Table 2).

When the pots were simultaneously treated with a

combination of purified auxinAH18 and auxinK11, a

synergistic effect was noted in the growth promotion of

roots, stems, and leaves of red-pepper and tomato plants

(Table 2).

To determined the synergistic effect of plant growth

promotion by the strains of B. subtilis AH18 and B.

licheniformis K11, 5×107 or 108 cells of either strain, and

both strains together, were used for pot experiments. All

combinations showed growth promotion activity. In

particular, the combination of B. subtilis AH18 (5×107

cells) and B. licheniformis K11 (5×107 cells) for a total of

108 cells synergistically promoted growth of stem and

leaves on red-pepper and tomato plants as compared to

the individual application of 108 cells of either bacterial

strain (Fig. 8).

The selected PGPR B. subtilis AH18 and B.

licheniformis K11 produce auxins (IAA:IBA:IPA=

1:1.5:2.6, IBA) as well as antifungal β-glucanase (55

kDa, 54 kDa), and siderophore (2,3-dihydroxybenzoyl-

glycine-threonine, 2,3-dihydroxybenzoyl-threonine) [Woo

et al., 2006; 2007; Woo and Kim, 2007; 2008].

Furthermore, B. licheniformis K11 could also produce an

antibiotic, iturin (unpublished observation) and both

strains are capable of solubilizing insoluble calcium

phosphate (unpublished observation, Table 3). The

variety of suppression mechanisms of PGPR strains

AH18 and K11 may be well-suited to their use as

biocontrol agents. It should be possible to develop

environmentally-friendly means of using these strains for

organic farming in pathogen-vulnerable conditions by

exploiting the combination of two Bacillus strains which

had a synergistic plant growth promotion and plant

diseases suppression ability.

Fig. 6. Comparison of seed germination ratio of variousplants by partially purified PGPAH18 and PGPK11. After 3days incubation of the seedings in Petri dishes, the numberof germinations was determined for each sample. Valuesare expressed as the means of three replicates, eachcontaining 40 seeds. Standard errors were determined atp≤0.05.

Fig. 7. Comparison of synergistic plant growth by the treatment of auxinAH18 and auxinK11 on red-pepper and tomato.The left panel shows red pepper plant and the right panel shows tomato plant. In each panel, the lanes are: C, only watertreatment; 1, auxinAH18 treatment; 2, auxinK11 treatment; 3, auxinAH18 and auxinK11 treatment. The purified auxins were treatedat concentrations of 100 ppm. The plants were watered every 5 days with 50 mL of sterile water. The pictures were takenat 20 days.


Acknowledgments. This research was supported by

the Yeungnam University research grants in 2009 and

Technology Development Program (107013-03) for

Agriculture and Forestry, Ministry for Agriculture,

Forestry and Fisheries, Republic of Korea.

References

Costacurta A and Vanderleyden J (1995) Synthesis of phyto-

hormones by plant-associated bacteria. Crit Rev Micro-

biol 21, 1-18.

Table 2. Effect of the partially purified auxinAH18 and auxinK11 on adventitious root formation and development inmungbean hypocotyl cutting

Sample

Number of mung-bean adventitious roots (ea)

Length ofmung-bean adventitious root (mm)

5 ppm 10 ppm 20 ppm 50 ppm 5 ppm 10 ppm 20 ppm 50 ppm

*IBA 08.0±0.9 10.6±1.0 2.9±0.1 0 3.9±0.3 4.6±0.2 0.8±0.1 0

*IAA 09.9±0.8 11.0±1.8 1.6±0.3 0 4.1±0.1 5.6±0.3 1.1±0.1 0

AuxinK11 11.8±0.8 14.3±0.1 0 0 5.9±0.2 6.1±0.1 0 0

AuxinAH18 12.0±0.8 16.6±1.3 0.8±0.8 0 6.0±0.1 6.6±0.2 0.3±0.1 0

AuxinAH18+AuxinK11 15.1±0.9 19.2±1.5 2.8±0.1 0 6.2±0.1 7.2±0.2 1.4±0.1 0

*Commercially-obtained IAA and IBA.

Values are expressed as the means of three replicates, each containing 20 plants.

Standard errors were determined at p≤0.05.

Fig. 8. Synergistic growth by mixed treatment of B. subtilis AH18 and B. licheniformis K11 cells on red-pepper andtomato. The left panel shows red pepper plant and the right panel shows tomato plant. In each panel, the lanes are: C,only water treatment; 1, treatment with B. subtilis AH18 (108 cells); 2, treatment with B. licheniformis K11 (108 cells); 3,treatment with a mixture of B. subtilis AH18 (5×107 cells) and B. licheniformis K11 (5×107 cells). The plants were wateredevery 5 days with 50 mL of sterile water. The pictures were taken at 25 days.

Table 3. Mechanisms of plant growth promotion by the selected PGPR strains

Functions B. subtilis AH18 B. licheniformis K11

Plant growthhormones

AuxinIAA (175 kDa)IBA (203 kDa)IPA (189 kDa)

IBA (203 kDa)

Antifungalactivity

Siderophore2,3-dihydroxybenzoyl

-glycine-threonine (883 Da)2,3-dihydroxybenzoic-threonine (808 Da)

β-1,4 glucanasefungal cell degrading cellulase

(55 kDa)fungal cell degrading cellulase

(54 kDa)

AntibioticIturin A gene

(1.5 kbp)

Phosphate solubilization(calcium phosphate hydrolase)

++ ++


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